Non-invasive methods for surgery in the vasculature

ABSTRACT

Novel methods of non-invasive intravascular surgery are disclosed.

REFERENCE TO COPENDING APPLICATIONS

[0001] This application is a divisional of copending application Ser.No. 09/613,210, filed Jul. 10, 2000, which in turn is a divisional ofapplication Ser. No. 08/476,317, filed Jun. 7, 1995, now U.S. Pat. No.6,088,613.

[0002] Application Ser. No. 08/476,317 is, in turn, acontinuation-in-part of copending application Ser. No. 08/401,974, filedMar. 9, 1995, now U.S. Pat. No. 5,922,304, which is acontinuation-in-part of copending application Ser. No. 08/212,553, filedMar. 11, 1994, now abandoned, the disclosures of which are herebyincorporated herein by reference, in their entirety, and priority towhich is hereby claimed.

[0003] Copending application Ser. No. 08/076,250, filed Jun. 11, 1993,now U.S. Pat. No. 5,580,575, which is a continuation-in-part of U.S.Ser. No. 716,899, now abandoned, and Ser. No. 717,084, now U.S. Pat. No.5,228,446, each filed Jun. 18, 1991, which in turn arecontinuations-in-part of U.S. Ser. No. 569,828, filed Aug. 20, 1990, nowU.S. Pat. No. 5,088,499, which in turn is a continuation-in-part of U.S.Ser. No. 455,707, filed Dec. 22, 1989, now abandoned, disclosestherapeutic drug delivery systems comprising gas filled micro spherescontaining a therapeutic agent, with particular emphasis on the use ofultrasound techniques to monitor and determine the presence of saidmicrospheres in a patient's body, and then to rupture said microspheresin order to release said therapeutic agent in the region of thepatient's body where said microspheres are found.

[0004] Copending application Ser. No. 08/076,239, filed Jun. 11, 1993,now U.S. Pat. No. 5,469,854, which has the identical parentage ofpreceding applications as Ser. No. 08/076,250 set out immediately above,discloses methods and apparatus for preparing gas filled microspheressuitable for use as contrast agents for ultrasonic imaging or as drugdelivery agents.

[0005] Copending application Ser. No. 08/307,305, filed Sep. 16, 1994,now U.S. Pat. No. 5,773,024, and copending application Ser. No.08/159,687, now U.S. Pat. No. 5,585,112, and Ser. No. 08/160,232, nowU.S. Pat. No. 5,542,935, both of which were filed Nov. 30, 1993, whichin turn are continuation-in-parts, respectively, of application Ser. No.08/076,239, now U.S. Pat. No. 5,469,854 and U.S. Ser. No. 08/076,250,now U.S. Pat. No. 5,580,575, both of which were filed Jun. 11, 1993,disclose novel therapeutic delivery systems and methods of preparing gasand gaseous precursor filled microspheres and multiphase lipid and gascompositions useful in diagnostic and therapeutic applications.

[0006] Benefit of the filing dates of application Ser. Nos. 08/307,305,08/159,687, 08/160,232, 08/076,239 and 08/076,250, and their parentage,is hereby claimed, and they are incorporated herein by reference intheir entirety.

[0007] Reference is also made to application Ser. No. 07/507,125, filedApr. 10, 1990, now abandoned, which discloses the use of biocompatiblepolymers, either alone or in admixture with one or more contrast agentssuch as paramagnetic, superparamagnetic or proton density contrastagents. The polymers or polymer/contrast agent admixtures may optionallybe admixed with one or more biocompatible gases to increase therelaxivity of the resultant preparation.

BACKGROUND OF THE INVENTION

[0008] 1. Field of the Invention

[0009] This invention relates to the field of magnetic resonanceimaging, more specifically to the use of stabilized gas filled vesiclesas contrast media for magnetic resonance imaging (MRI) directedultrasound surgery.

[0010] There are a variety of imaging techniques that have been used todiagnose disease in humans. One of the first imaging techniques employedwas X-rays. In X-rays, the images produced of the patients' body reflectthe different densities of body structures. To improve the diagnosticutility of this imaging technique, contrast agents are employed toincrease the density of tissues of interest as compared to surroundingtissues to make the tissues of interest more visible on X-ray. Bariumand iodinated contrast media, for example, are used extensively forX-ray gastrointestinal studies to visualize the esophagus, stomach,intestines and rectum. Likewise, these contrast agents are used forX-ray computed tomographic studies (that is, computer assistedtomography or CAT) to improve visualization of the gastrointestinaltract and to provide, for example, a contrast between the tract and thestructures adjacent to it, such as the vessels or lymph nodes. Suchcontrast agents permit one to increase the density inside the esophagus,stomach, intestines and rectum, and allow differentiation of thegastrointestinal system from surrounding structures.

[0011] Magnetic resonance imaging (MRI) is a relatively new imagingtechnique which, unlike X-rays, does not utilize ionizing radiation.Like computer assisted tomography (CAT), MRI can make cross-sectionalimages of the body, however MRI has the additional advantage of beingable to make images in any scan plane (i.e., axial, coronal, sagittal ororthogonal). Unfortunately, the full utility of MRI as a diagnosticmodality for the body is hampered by the need for new or better contrastagents. Without suitable agents, it is often difficult using MRI todifferentiate the target tissue from adjacent tissues. If bettercontrast agents were available, the overall usefulness of MRI as animaging tool would improve, and the diagnostic accuracy of this modalitywould be greatly enhanced.

[0012] MRI employs a magnetic field, radio frequency energy and magneticfield gradients to make images of the body. The contrast or signalintensity differences between tissues mainly reflect the T1(longitudinal) and T2 (transverse) relaxation values and the protondensity (effectively, the free water content) of the tissues. Inchanging the signal intensity in a region of a patient by the use of acontrast medium, several possible approaches are available. For example,a contrast medium could be designed to change either the T1, the T2 orthe proton density.

[0013] 2. Brief Description of the Prior Art

[0014] In the past, attention has mainly been focused on paramagneticcontrast media for MRI. Paramagnetic contrast agents contain unpairedelectrons which act as small local magnets within the main magneticfield to increase the rate of longitudinal (T1) and transverse (T2)relaxation. Most paramagnetic contrast agents are metal ions which inmost cases are toxic. In order to decrease toxicity, these metal ionsare generally chelated using ligands. The resultant paramagnetic metalion complexes have decreased toxicity.

[0015] Metal oxides, most notably iron oxides, have also been tested asMRI contrast agents. While small particles of iron oxide, e.g., under 20nm diameter, may have paramagnetic relaxation properties, theirpredominant effect is through bulk susceptibility. Therefore magneticparticles have their predominant effect on T2 relaxation. Nitroxides areanother class of MRI contrast agent which are also paramagnetic. Thesehave relatively low relaxivity and are generally less effective thanparamagnetic ions as MRI contrast agents. All of these contrast agentscan suffer from some toxic effects in certain use contexts and none ofthem are ideal for use as perfusion contrast agents by themselves.

[0016] The existing MRI contrast agents suffer from a number oflimitations. For example, positive contrast agents are known to exhibitincreased image noise arising from intrinsic peristaltic motions andmotions from respiration or cardiovascular action. Positive contrastagents such as Gd-DTPA are subject to the further complication that thesignal intensity depends upon the concentration of the agent as well asthe pulse sequence used. Absorption of contrast agent from thegastrointestinal tract, for example, complicates interpretation of theimages, particularly in the distal portion of the small intestine,unless sufficiently high concentrations of the paramagnetic species areused (Kornmesser et al., Magn. Reson. Imaging, 6:124 (1988)). Negativecontrast agents, by comparison, are less sensitive to variation in pulsesequence and provide more consistent contrast. However at highconcentrations, particulates such as ferrites can cause magneticsusceptibility artifacts which are particularly evident, for example, inthe colon where the absorption of intestinal fluid occurs and thesuperparamagnetic material may be concentrated. Negative contrast agentstypically exhibit superior contrast to fat, however on T1-weightedimages, positive contrast agents exhibit superior contrast versus normaltissue. Since most pathological tissues exhibit longer T1 and T2 thannormal tissue, they will appear dark on T1-weighted and bright onT2-weighted images. This would indicate that an ideal contrast agentshould appear bright on T1-weighted images and dark on T2-weightedimages. Many of the currently available MRI contrast media fail to meetthese dual criteria.

[0017] Toxicity is another problem with the existing contrast agents.With any drug there is some toxicity, the toxicity generally being doserelated. With the ferrites there are often symptoms of nausea after oraladministration, as well as flatulence and a transient rise in serumiron. The paramagnetic contrast agent Gd-DTPA is an organometalliccomplex of gadolinium coupled with the complexing agent diethylenetriamine pentaacetic acid. Without coupling, the free gadolinium ion ishighly toxic. Furthermore, the peculiarities of the gastrointestinaltract, for example, wherein the stomach secretes acids and theintestines release alkalines, raise the possibility of decoupling andseparation of the free gadolinium or other paramagnetic agent from thecomplex as a result of these changes in pH during gastrointestinal use.Certainly, minimizing the dose of paramagnetic agents is important forminimizing any potential toxic effects.

[0018] New and/or better contrast agents useful in magnetic resonanceimaging as well as improved imaging techniques are needed. The presentinvention is directed, inter alia, to these important ends.

[0019] In the work on MRI contrast agents described above forapplication Ser. No. 07/507,125, filed Apr. 10, 1990, it has beendisclosed how gas can be used in combination with polymer compositionsand paramagnetic or superparamagnetic agents as MRI contrast agents.Therein it has been shown how the gas stabilized by said polymers wouldfunction as an effective susceptibility contrast agent to decreasesignal intensity on T2 weighted images; and that such systems areparticularly effective for use as gastrointestinal MRI contrast media.

[0020] Widder et al. published application EP-A-0 324 938 disclosesstabilized microbubble-type ultrasonic imaging agents produced fromheat-denaturable biocompatible protein, e.g., albumin, hemoglobin, andcollagen.

[0021] There is also mentioned a presentation believed to have been madeby Moseley et al., at a 1991 Napa, California meeting of the Society forMagnetic Resonance in Medicine, which is summarized in an abstractentitled “Microbubbles: A Novel MR Susceptibility Contrast Agent.” Themicrobubbles which are utilized comprise air coated with a shell ofhuman albumin. The stabilized gas filled vesicles of the presentinvention are not suggested.

[0022] For intravascular use, however, the inventors have found that foroptimal results, it is advantageous that any gas be stabilized withflexible compounds. Proteins such as albumin may be used to stabilizethe bubbles but the resulting bubble shells may be brittle andinflexible. This is undesirable for several reasons. Firstly, a brittlecoating limits the capability of the bubble to expand and collapse. Asthe bubble encounters different pressure regions within the body (e.g.,moving from the venous system into the arteries upon circulation throughthe heart) a brittle shell may break and the gas will be lost. Thislimits the effective period of time for which useful contrast can beobtained in vivo from the bubbles contrast agents. Also such brittle,broken fragments can be potentially toxic. Additionally the inflexiblenature of brittle coatings such as albumin, and stiff resulting bubblesmake it extremely difficult to measure pressure in vivo.

[0023] Quay published application WO 93/05819 discloses that gases withhigh Q numbers are ideal for forming stable gases, but the disclosure islimited to stable gases, rather than their stabilization andencapsulation, as in the present invention. In a preferred embodimentdescribed on page 31, sorbitol is used to increase viscosity, which inturn is said to extend the life of a microbubble in solution. Also, itis not an essential requirement of the present invention that the gasinvolved have a certain Q number or diffusibility factor.

[0024] Lanza et al. published application WO 93/20802 disclosesacoustically reflective oligolamellar liposomes, which are multilamellarliposomes with increased aqueous space between bilayers or haveliposomes nested within bilayers in a nonconcentric fashion, and thuscontain internally separated bilayers. Their use as ultrasonic contrastagents to enhance ultrasonic imaging, and in monitoring a drug deliveredin a liposome administered to a patient, is also described.

[0025] D'Arrigo U.S. Pat. Nos. 4,684,479 and 5,215,680 disclosegas-in-liquid emulsions and lipid-coated microbubbles, respectively.

[0026] In accordance with the present invention it has been discoveredthat stabilized gas filled vesicles are extremely effective, non-toxiccontrast agents for simultaneous magnetic resonance focused noninvasiveultrasound.

SUMMARY OF THE INVENTION

[0027] The present invention is directed to a method of magneticresonance imaging focused surgical and therapeutic ultrasound comprisingadministering a contrast medium for magnetic resonance imagingcomprising gas filled vesicles to a patient requiring surgery, usingsaid contrast medium to scan the patient with magnetic resonance imagingto identify the region of the patient requiring surgery, and applyingultrasound to the region to carry out surgery. The application ofultrasound may be followed by a second scanning step whereby the patientis scanned with magnetic resonance imaging. The ultrasound applicationmay be simultaneous with magnetic resonance imaging. The scanning andsurgical ultrasound steps may be performed repeatedly until the desiredeffect is achieved. The gas filled vesicles may comprise a therapeuticwhich may be released to a localized region of a patient uponultrasound.

[0028] In addition, the present invention comprises a method for thecontrolled delivery of a therapeutic to a region of a patient usingmagnetic resonance imaging focused therapeutic ultrasound comprisingadministering to the patient vesicles comprising gas-filled vesiclescomprising a therapeutic compound; monitoring the vesicles usingmagnetic resonance imaging to determine the presence of the vesicles inthe region; and rupturing the vesicles using ultrasound to release thetherapeutic in the region.

[0029] The present invention is also directed to a method of magneticresonance focused surgical ultrasound comprising administering acontrast medium for magnetic resonance imaging comprising gaseousprecursor filled vesicles to a patient requiring surgery, allowing thegaseous precursor to undergo a phase transition from a liquid to a gas,scanning the patient with magnetic resonance imaging to identify theregion of the patient requiring surgery, and applying surgicalultrasound to the region. The phase transition step and the magneticresonance scanning step may be performed simultaneously.

[0030] The contrast medium comprises stabilized gas filled vesicles,wherein the gas is a biocompatible gas, e.g., nitrogen orperfluoro-propane, but may also be derived from a gaseous precursor,e.g., perfluorooctylbromide, and the vesicles are stabilized by beingformed from a stabilizing compound, e.g., a biocompatible lipid orpolymer. The present invention may be carried out, often withconsiderable attendant advantage, by using gaseous precursors to formthe gas of the gas filled vesicles. These gaseous precursors may beactivated by a number of factors, but preferably are temperatureactivated. Such a gaseous precursor is a compound which, at a selectedactivation or transition temperature, changes phases from a liquid orsolid to-a gas. Activation thus takes place by increasing thetemperature of the compound from a point below, to a point above theactivation or transition temperature. Where a lipid is used to form thevesicle, the lipid may be in the form of a monolayer or bilayer, and themono- or bilayer lipids may be used to form a series of concentric mono-or bilayers. Thus, the lipid may be used to form a unilamellar liposome(comprised of one monolayer or bilayer lipid), an oligolamellar liposome(comprised of two or three monolayer or bilayer lipids) or amultilamellar liposome (comprised of more than three monolayer orbilayer lipids). Preferably, the biocompatible lipid comprises aphospholipid. Optionally, the contrast medium may include paramagneticand/or superparamagnetic contrast agents, preferably encapsulated by thevesicles. Also, optionally, the contrast medium may further comprise aliquid fluorocarbon compound, e.g., a perfluorocarbon, to furtherstabilize the vesicles. Preferably the fluorocarbon liquid isencapsulated by the vesicles.

[0031] These and other aspects of the invention will become moreapparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention is directed, inter alia, to a method ofmagnetic resonance imaging focused surgical and therapeutic ultrasoundcomprising administering a contrast medium for magnetic resonanceimaging comprising gas filled vesicles to a patient requiring surgery,using contrast medium to scan the patient with magnetic resonanceimaging to identify the region of the patient requiring surgery, andapplying ultrasound to the region to carry out surgery. The ultrasoundstep may be performed simultaneously with magnetic resonance imaging.The application of ultrasound may be followed by a second scanning stepwhereby the patient is scanned with magnetic resonance imaging. The gasfilled vesicles may comprise a therapeutic which may be released to alocalized region of a patient upon ultrasound.

[0033] The scanning and surgical ultrasound steps may be performedrepeatedly until the desired effect is achieved. In accordance with thepresent invention, simultaneous refers to scanning with ultrasound andmagnetic resonance concurrently or synchronously; sequentially orsuccessively; such that visualization of the disruption of vesicles andtissues by ultrasound is observed. Thus, ultrasound and magneticresonance may be performed at the same time, or one may be followed bythe other. The use of magnetic resonance imaging together withultrasound improves the accuracy of currently available imagingmodalities. The precision of magnetic resonance imaging and ultrasoundtogether confirm the location of the vesicles, as the entire body isable to be scanned by magnetic resonance imaging which provides a largefield of view, and, once located, the vesicles may be ruptured byultrasound in the given regions of the body.

[0034] The present invention is also directed to a method of magneticresonance focused surgical ultrasound comprising administering acontrast medium for magnetic resonance imaging comprising gaseousprecursor filled vesicles to a patient requiring surgery, allowing thegaseous precursor to undergo a phase transition from a liquid to a gas,using said contrast medium to scan the patient with magnetic resonanceimaging to identify the region of the patient requiring surgery, andapplying surgical ultrasound to the region.

[0035] In addition, the present invention comprises a method for thecontrolled delivery of a therapeutic to a region of a patient usingmagnetic resonance focused therapeutic ultrasound comprisingadministering to the patient contrast medium comprising gas-filledvesicles comprising a therapeutic compound; monitoring the vesiclesusing magnetic resonance imaging to determine the presence of thevesicles in the region; and rupturing the vesicles using ultrasound torelease the therapeutic in the region.

[0036] As employed above and throughout the disclosure, the followingterms, unless otherwise indicated, shall be understood to have thefollowing meanings.

[0037] “Magnetic resonance imaging” (MRI) uses a static main magneticfield; pulsed radiofrequency energy and pulsed magnetic gradients tocreate images, i.e. to visualize the vesicles. The radiofrequency andelectrical gradients may be used to cause local energy deposition andactivate the vesicles, however, ultrasound is the preferred energy forthe purpose of activating the vesicles. In carrying out the magneticresonance imaging method of the present invention, the contrast mediumcan be used alone, or in combination with other diagnostic, therapeuticor other agents. Such other agents include excipients such as flavoringor coloring materials. The magnetic resonance imaging techniques whichare employed are conventional and are described, for example, in D. M.Kean and M. A. Smith, Magnetic Resonance Imaging: Principles andApplications, (William and Wilkins, Baltimore 1986). Contemplated MRItechniques include, but are not limited to, nuclear magnetic resonance(NMR) and electronic spin resonance (ESR), and magnetic resonanceangioplasty (MRA). The preferred imaging modality is NMR. Of course, inaddition to MRI, magnetic imaging may also be used to detect vesicleswithin the scope of the present invention. Magnetic imaging uses amagnetic field yet need not use pulsed gradients or radiofrequencyenergy. Magnetic imaging may be used to detect magnetic vesicles, suchas and not limited to ferromagnetic vesicles. Magnetic imaging may beperformed by a magnetometer superconducting quantum inferometry device(SQUID). SQUID permits rapid screening of all of the body tissues forthe magnetic particles; the ultrasound may then be localized to thoseregions. In this application, magnetic resonance imaging includesmagnetic imaging, while it is understood that magnetic imaging is theimaging of magnetic vesicles and does not include resonance of thenuclei thereof.

[0038] “Ultrasound imaging” is performed on the tissues of interest andultrasound energy may be used to activate or rupture the vesicles oncethey reach their intended tissue destination. Focused or directedultrasound refers to the application of ultrasound energy to aparticular region of the body, such that the ultrasound energy isconcentrated to a selected area or target zone. In addition, focusedrefers to the magnetic resonance which guides the ultrasound byvisualizing the vesicles and the target zone; and simultaneous withultrasound visualizing the disruption of tissues thereby. Noninvasiverefers to the disruption or disturbance of internal body tissues withoutan incision in the skin. Ultrasound, as defined in accordance with thepresent invention refers to surgery resulting in tissue necrosis, i.e.disruption or destruction of tissue; repair of apertures, openings,breaks, or tears in tissue (such as a hernia); alleviation of all orpart of diseased tissue (such as tumors); and the activation or ruptureof vesicles adjacent to tissue by ultrasonic energy. Ultrasound is adiagnostic imaging technique which is unlike nuclear medicine and X-rayssince it does not expose the patient to the harmful effects of ionizingradiation. Moreover, unlike magnetic resonance imaging, ultrasound isrelatively inexpensive and can be conducted as a portable examination.In using the ultrasound technique, sound is transmitted into a patientor animal via a transducer. When the sound waves propagate through thebody, they encounter interfaces from tissues and fluids. Depending onthe acoustic properties of the tissues and fluids in the body, theultrasound sound waves are partially or wholly reflected or absorbed.When sound waves are reflected by an interface they are detected by thereceiver in the transducer and processed to form an image. The acousticproperties of the tissues and fluids within the body determine thecontrast which appears in the resultant image. Alternatively, ultrasoundmay be used to visualize the vesicles and magnetic resonance imaging maybe used to activate the vesicles. In addition, the strength ofultrasound energy may be at an intensity to result in rupture oractivation of vesicles. The activation of the vesicles in turn disruptsthe adjacent tissue such that necrosis of the tissue results.

[0039] Any of the various types of diagnostic ultrasound imaging devicesmay be employed in the practice of the invention, the particular type ormodel of the device not being critical to the method of the invention.Also suitable are devices designed for administering ultrasonichyperthermia, such devices being described in U.S. Pat. Nos. 4,620,546,4,658,828, and 4,586,512, the disclosures of each of which are herebyincorporated herein by reference in their entirety. Preferably, thedevice employs a resonant frequency (RF) spectral analyzer. Thetransducer probes may be applied externally or may be implanted.Ultrasound is generally initiated at lower intensity and duration,preferably at peak resonant frequency, and then intensity, time, and/orresonant frequency increased until the microsphere ruptures.

[0040] “Vesicle” refers to a spherical entity which is characterized bythe presence of an internal void. Preferred vesicles are formulated fromlipids, including the various lipids described herein. In any givenvesicle, the lipids may be in the form of a monolayer or bilayer, andthe mono- or bilayer lipids may be used to form one or more mono- orbilayers. In the case of more than one mono- or bilayer, the mono- orbilayers are generally concentric. The vesicles described herein includesuch entities commonly referred to as liposomes, micelles, bubbles,microbubbles, aerogels, clathrate bound vesicles, and the like. Thus,the lipids may be used to form a unilamellar vesicle (comprised of onemonolayer or bilayer), an oligolamellar vesicle (comprised of about twoor about three monolayers or bilayers) or a multilamellar vesicle(comprised of more than about three monolayers or bilayers). Theinternal void of the vesicles may be filled with a liquid, including,for example, an aqueous liquid, a gas, a gaseous precursor, and/or asolid or solute material, including, for example, a targeting ligandand/or a bioactive agent, as desired.

[0041] “Liposome” refers to a generally spherical cluster or aggregateof amphipathic compounds, including lipid compounds, typically in theform of one or more concentric layers. Most preferably the gas filledliposome is constructed of a single layer (i.e. unilamellar) or a singlemonolayer of lipid. A wide variety of lipids may be used to fabricatethe liposomes including phospholipids and non-ionic surfactants (e.g.niosomes). Most preferably the lipids comprising the gas filledliposomes are in the gel state at physiological temperature. Theliposomes may be cross-linked or polymerized and may bear polymers suchas polyethylene glycol on their surfaces. Targeting ligands directed toendothelial cells are bound to the surface of the gas filled liposomes.A targeting ligand is a substance which is bound to a vesicle anddirects the vesicle to a particular cell type such as and not limited toendothelial tissue and/or cells. The targeting ligand may be bound tothe vesicle by covalent or non-covalent bonds. The liposomes may also bereferred to herein as lipid vesicles. Most preferably the liposomes aresubstantially devoid of water in their interiors.

[0042] “Micelle” refers to colloidal entities which form from lipidiccompounds when the concentration of the lipidic compounds, such aslauryl sulfate, is above a critical concentration. Since many of thecompounds which form micelles also have surfactant properties (i.e.ability to lower surface tension and both water and fatloving—hydrophilic and lipophilic domains), these same materials mayalso be used to stabilize bubbles. In general these micellular materialsprefer to adopt a monolayer or hexagonal H2 phase configuration, yet mayalso adopt a bilayer configuration. When a micellular material is usedto form a gas filled vesicle, the compounds will generally adopt aradial configuration with the aliphatic (fat loving) moieties orientedtoward the vesicle and the hydrophilic domains oriented away from thevesicle surface. For targeting to endothelial cells, the targetingligands may be attached to the micellular compounds or to amphipathicmaterials admixed with the micellular compounds. Alternatively,targeting ligands may be adsorbed to the surface of the micellularmaterials stabilizing the vesicles.

[0043] “Aerogel” refers to structures which are similar to microspheresexcept that the internal structure of the aerogels is generallycomprised of multiple small voids rather than one void. Additionally theaerogels are preferably constructed of synthetic materials (e.g. a foamprepared from baking resorcinol and formaldehyde), however naturalmaterials such as polysaccharides or proteins may also be used toprepare aerogels. Targeting ligands may be attached to the surface ofthe aerogel.

[0044] “Clathrates” are generally solid materials which bind thevesicles as a host rather than coating the surface of the vesicle. Asolid, semi-porous, or porous clathrate may serve as the agentstabilizing the vesicle, however the clathrate itself does not coat theentire surface of the vesicle. Rather, the clathrate forms a structureknown as a “cage” having spaces into which the vesicles may fit. One ormore vesicles may be adsorbed by the clathrate. Similar to microspheres,one or more surfactants may be incorporated with the clathrate and thesesurfactants will help to stabilize the vesicle. The surfactants willgenerally coat the vesicle and help to maintain the association of thevesicle with the clathrate. Useful clathrate materials for stabilizingvesicles include porous apatites such as calcium hydroxyapatite andprecipitates of polymers with metal ions such as alginic acid withcalcium salts. Targeting ligands directed to endothelial cells may beincorporated into the clathrate itself or into the surfactant materialused in association with the clathrate.

[0045] While not intending to be bound by any particular theory ofoperation, the present invention is believed to rely, at least in part,on the fact that gas, liquid, and solid phases have different magneticsusceptibilities. At the interface of gas and water, for example, themagnetic domains are altered and this results in dephasing of the spinsof, e.g., the hydrogen nuclei. In imaging, this is seen as a decrease insignal intensity adjacent to the gas/water interface. This effect ismore marked on T2 weighted images and most prominent on gradient echopulse sequences. The effect is increased by using narrow bandwidthextended read-out pulse sequences. The longer the echo time on agradient echo pulse sequence, the greater the effect (i.e., the greaterthe degree and size of signal loss).

[0046] The stabilized gas filled vesicles useful in the presentinvention are believed to rely on this phase magnetic susceptibilitydifference, as well as on the other characteristics described in moredetail herein, to provide high performance level magnetic resonanceimaging contrast medium and effective rupture of vesicles of thecontrast medium. The vesicles are formed from, i.e., created out of, amatrix of stabilizing compounds which permit the gas filled vesicles tobe established and thereafter retain their size and shape for the periodof time required to be useful in magnetic resonance imaging. Thecompounds also permit rupture of the vesicles at a certain energy level,which energy is preferably ultrasound energy. These stabilizingcompounds are most typically those which have a hydrophobic/hydrophiliccharacter which allows them to form monolayers or bilayers, etc., andvesicles, in the presence of water. Thus, water, saline or some otherwater-based medium, often referred to hereafter as a diluent, isgenerally an aspect of the stabilized gas filled vesicle contrast mediumof the present invention.

[0047] The stabilizing compound may, in fact, be a mixture of compoundswhich contribute various desirable attributes to the stabilizedvesicles. For example, compounds which assist in the dissolution ordispersion of the fundamental stabilizing compound have been foundadvantageous. A further element of the stabilized vesicles is a gas,which can be a gas at the time the vesicles are made, or can be agaseous precursor which, responsive to an activating factor, such astemperature, is transformed from the liquid or solid phase to the gasphase. The various aspects of the stabilized gas filled contrast mediumuseful in the present invention will now be described.

[0048] Methods of Use

[0049] In accordance with the present invention there is provided amethod of simultaneous magnetic resonance focused noninvasiveultrasound. The imaging process of the present invention may be carriedout by administering a contrast medium for magnetic resonance imagingcomprising gas filled vesicles to a patient requiring surgery, scanningthe patient with magnetic resonance imaging to identify the region ofthe patient requiring surgery, and simultaneously applying ultrasoundand magnetic resonance to the region. By region of a patient, it ismeant the whole patient or a particular area or portion of the patient.

[0050] After administration to a patient, the vesicles, which arevisible on magnetic resonance imaging, are visualized by MRI. When thelocation of the vesicles is determined to be in the desired region ofthe patient, as ascertained by MRI, then energy, preferably ultrasoundenergy, is applied to the region. The vesicles are activated by theenergy, heating and direct and rapid coagulative necrosis of thesurrounding tissue (i.e. surgical ultrasound) results. Simultaneously,the region may also be visualized by magnetic resonance imaging ifdesired. Preferably, the energy used for vesicle activation is highenergy continuous wave ultrasound, preferably over 50 milliwatts/cm²,even more preferably over 100 milliwatts/cm². Depending upon the desiredtherapeutic effect, the energy may be even higher, up to about 10watts/cm². Most preferably, the energy is deposited into the tissuesusing a hand held magnetic resonance compatible ultrasound transducer.The ultrasound transducer is made out of non-ferrous andnon-ferromagnetic material. The cables supplying energy to theultrasound transducers may have Faraday shields to decrease thepotential for artifacts which may be caused by the electrical energypassing through the cables to supply the transducers.

[0051] The amount of energy and pulse duration of ultrasound used fortherapy will vary depending upon the therapeutic purpose. The ultrasonicenergy is preferably focused and the focal zone is chosen to target thedesired regions of vesicles.

[0052] Focused ultrasonic surgery may be performed at energies of about2 watts/cm². Focused ultrasonic surgery energy may be at least 2watts/cm² to about 10 watts/cm². Direct and rapid coagulative necrosisof the tissue results. Simultaneous MRI may be performed with thevesicles used to visualize the target zone or region. Then together withultrasound, the vesicles potentiate the surgery in the target zone.

[0053] Energy range of from about 500 mW/cm² to about 10 watts/cm²,preferably greater than about 1 watt is useful for cavitational tissuedestruction. The vesicles lower the cavitation threshold such thatcavitation will occur within the target tissues at a low energythreshold resulting in tissue destruction.

[0054] Rupture or activation of vesicles may take place at an energyrange of from about 50 mW/cm² to about 500 mW/cm². Vesicles may beruptured by non-cavitational interaction. As the vesicle is pulsedrapidly and strongly enough by ultrasound energy, the vesicle membranedegenerates. While there is likely a transient microdomain of increasedtemperature associated with the vesicle rupture, this process may notdamage the surrounding tissues when energy and pulsing is applied at anindicated energy range. This effect of vesicle rupture may beadvantageously used for localized delivery of a therapeutic. Thus, atherapeutic may be released to a region of the body with this technique.Further, energy from vesicle rupture may be used to create shock wavesso that the therapeutic is also deposited released to adjacent tissues.This is particularly useful with gene therapy wherein the shock wavesmay be used to open transient pores in adjacent cell membranes andfacilitate cellular uptake of genetic material.

[0055] An energy range of about 500 mW/cm² to about 5 watts/cm², withvesicles as nuclei, may be used to increase the conversion of highenergy sound into localized tissue, thereby heating the tissue andinducing hyperthermia.

[0056] In the case of a gaseous precursor, as ultrasound energy isfocused on the precursor, it causes the precursor to convert to thegaseous state. The enlarging gaseous void creates a domain of increasingmagnetic susceptibility and is readily monitored on the magneticresonance images. Monitoring is particularly enhanced by selectingprecursors with well defined liquid to gas conversion temperatures, suchas perfluorohexane at 56° C.

[0057] The invention thus may also be used for non-invasive temperaturemonitoring during MRI. As the vesicles form from gaseous precursors, thematerials surrounding the gaseous precursor may be ruptured. Inaddition, a therapeutic may be released locally into the adjacent tissuewhere a therapeutic is co-entrapped within the vesicle. As the vesicleforms, the absorption of energy by the vesicle interface increases. Thismay be used to increase heating for hyperthermia as well as to rupturethe vesicle.

[0058] The contrast medium may be particularly useful in providingimages of and permitting ultrasound mediated surgery and/or drugdelivery in the cardiovascular region or the gastrointestinal region,but can also be employed more broadly such as in imaging the vasculatureor in other ways as will be readily apparent to those skilled in theart. Cardiovascular region, as that phrase is used herein, denotes theregion of the patient defined by the heart and the vasculature leadingdirectly to and from the heart. The phrase gastrointestinal region orgastrointestinal tract, as used herein, includes the region of a patientdefined by the esophagus, stomach, small and large intestines andrectum. The phrase vasculature, as used herein, denotes the bloodvessels (arteries, veins, etc.) in the body or in an organ or part ofthe body. The patient can be any type of mammal, but most preferably isa human.

[0059] The novel stabilized gas filled vesicles, useful as contrastmedium in simultaneous magnetic resonance focused noninvasiveultrasound, will be found to be suitable for use in all areas where MRIis employed.

[0060] As one skilled in the art would recognize, administration of thestabilized gas filled vesicles used in the present invention may becarried out in various fashions, such as intravascularly, orally,rectally, etc., using a variety of dosage forms. When the region to bescanned is the cardiovascular region, administration of the contrastmedium of the invention is preferably carried out intravascularly. Whenthe region to be scanned is the gastrointestinal region, administrationof the contrast medium of the invention is preferably carried out orallyor rectally. The useful dosage to be administered and the particularmode of administration will vary depending upon the age, weight and theparticular mammal and region thereof to be scanned, and the particularcontrast medium of the invention to be employed. Typically, dosage isinitiated at lower levels and increased until the desired contrastenhancement is achieved. Various combinations of the stabilized gasfilled vesicles may be used to modify the relaxation behavior of themedium or to alter properties such as the viscosity, osmolarity orpalatability (in the case of orally administered materials). In carryingout the simultaneous magnetic resonance focused noninvasive ultrasoundmethod of the present invention, the contrast medium can be used alone,or in combination with other diagnostic, therapeutic or other agents.Such other agents include excipients such as flavoring or coloringmaterials. The magnetic resonance imaging techniques which are employedare conventional and are described, for example, in D. M. Kean and M. A.Smith, Magnetic Resonance Imaging: Principles and Applications, (Williamand Wilkins, Baltimore 1986). Contemplated MRI techniques include, butare not limited to, nuclear magnetic resonance (NMR) and electronic spinresonance (ESR). The preferred imaging modality is NMR.

[0061] As noted above, the routes of administration and areas ofusefulness of the gas filled vesicles are not limited merely to theblood volume space, i.e., the vasculature. Simultaneous magneticresonance focused noninvasive ultrasound can be achieved with the gasfilled vesicles used in the present invention if the vesicles areingested by mouth so as to image the gastrointestinal tract and rupturethe vesicles therein. Alternatively, rectal administration of thesestabilized gas vesicles can result in excellent imaging of the lowergastrointestinal tract including the rectum, descending colon,transverse colon, and ascending colon as well as the appendix, andrupturing the vesicles therein. It may be possible to achieve imaging ofthe jejunum and conceivably the ileum via this rectal route; and torupture the vesicles in these areas. As well, direct intraperitonealadministration may be achieved to visualize the peritoneum, and rupturethe vesicles therein. It is also contemplated that the stabilized gasvesicles may be administered directly into the ear canals such that onecan visualize the canals as well as the Eustachian tubes and, if aperforation exists, the inner ear. Further, activation or rupture of thevesicles in the ear may also take place. It is also contemplated thatthe stabilized gas vesicles may be administered intranasally to aid inthe visualization of the nasal septum as well as the nasal sinuses, andrupture of the vesicles therein. Interstitial administration is alsopossible. Other routes of administration of the vesicle contrast agentsof the present invention, and tissue areas whose imaging and rupture ofthe vesicles is enhanced thereby include, but are not limited to 1)intranasally for imaging the nasal passages and sinuses including thenasal region and sinuses and sinusoids; 2) intranasally and orally forimaging the remainder of the respiratory tract, including the trachea,bronchus, bronchioles, and lungs; 3) intracochlearly for imaging thehearing passages and Eustachian tubes, tympanic membranes and outer andinner ear and ear canals; 4) intraocularly for imaging the regionsassociated with vision; 5) intraperitoneally to visualize theperitoneum; and 6) intravesicularly, i.e., through the bladder, to imageall regions of the genitourinary tract via the areas thereof, including,but not limited to, the urethra, bladder, ureters, kidneys and renalvasculature and beyond, e.g., to perform cystography or to confirm thepresence of ureteral reflux. In addition, the brain, spine, pulmonayregion, and soft tissues such as and not including adipose tissue,muscle, and organs may be similarly imaged and surgery of these areasmay be achieved by ultrasound.

[0062] Use of the procedures of the invention permit ultrasound mediatedsurgery. By ultrasound mediated surgery it is meant surgery effectivelycausing tissue necrosis, i.e. disruption, destruction, or repair oftissue, such as repair of small tears (apertures, openings, or breaks)in tissue membranes (such as a hernia); alleviation of all or part ofdiseased tissue (such as tumors); and the activation or rupture ofvesicles adjacent to tissue by ultrasonic energy.

[0063] Gases and Gaseous Precursors

[0064] The vesicles of the invention encapsulate a gas and/or gaseousprecursor. The term “gas filled and/or gaseous precursor filled”, asused herein, means that the vesicles to which the present invention isdirected, have an interior volume that is comprised of at least about10% gas and gaseous precursor, preferably at least about 25% gas andgaseous precursor, more preferably at least about 50% gas and gaseousprecursor, even more preferably at least about 75% gas and gaseousprecursor, and most preferably at least about 90% gas and gaseousprecursor. In use, where the presence of gas is important, it ispreferred that the interior vesicle volume comprise at least about 10%gas, preferably at least about 25%, 50%, 75%, and most preferably atleast about 90% gas.

[0065] Any of the various biocompatible gases and gaseous precursors maybe employed in the gas and gaseous precursor filled vesicles of thepresent invention. Such gases include, for example, air, nitrogen,carbon dioxide, oxygen, argon, fluorine, xenon, neon, helium, rubidiumenhanced (hyperpolarized) xenon, rubidium enhanced argon, rubidiumenhanced helium, and rubidium enhanced neon, or any and all combinationsthereof. Of such gases, nitrogen and fluorine are preferred. Forexample, the use of NMR together with ¹⁹F provides more sensitivevisualization than the use of a liquid or a solid. Likewise, variousfluorinated gaseous compounds, such as various perfluorocarbon,hydrofluorocarbon, and sulfur hexafluoride gases may be utilized in thepreparation of the gas filled vesicles. Also, the gases discussed inQuay, published application WO 93/05819, including the high “Q” factorgases described therein, the disclosures of which are herebyincorporated herein by reference in their entirety, may be employed.Further, paramagnetic gases or gases such as ¹⁷O may be used. The oxygenshould be stabilized, since oxygen gas is soluble in blood.Stabilization may be accomplished by an impermeable shell, preferably ofa polymerized or cross-linked liposome or a cyanoacrylate microsphere;or used together with a perfluorocarbon, such as perfluoropentane orperfluorobutane. Of all of the gases, perfluorocarbons and sulfurhexafluoride are preferred. Suitable perfluorocarbon gases include, forexample, perfluorobutane, perfluorocyclobutane, perfluoromethane,perfluoroethane, perfluoropropane, and perfluoropentane,perfluorohexane, most preferably perfluoropropane. Also preferred are amixture of different types of gases, such as a perfluorocarbon gas andanother type of gas such as oxygen, etc. Indeed, it is believed that acombination of gases may be particularly useful in simultaneous magneticresonance focused noninvasive ultrasound applications.

[0066] The gaseous precursors may also be in the form of a solid. Sodiumbicarbonate crystals produce carbon dioxide gas upon activation of thesolid precursor form. Solid and liquid gaseous precursors areparticularly useful in ultrasonic hyperthermia which activates theprecursor into the gaseous state.

[0067] Notwithstanding the requirement that the gas and gaseousprecursor filled vesicles be made from stabilizing compounds, it ispreferred that a rather highly stable gas be utilized as well. By highlystable gas is meant a gas selected from those gases which will have low(limited) solubility and diffusability in aqueous media. Gases such asperfluorocarbons are less diffusible and relatively insoluble and assuch are easier to stabilize into the form of bubbles in aqueous media.

[0068] The use of gaseous precursors is an optional embodiment of thepresent invention. In particular, perfluorocarbons have been found to besuitable for use as gaseous precursors. As the artisan will appreciate,a given perfluorocarbon may be used as a gaseous precursor, i.e., in theliquid or solid state when the vesicles of the present invention arefirst made, or may be used as a gas directly, i.e., in the gas state, tomake the gas and gaseous precursor filled vesicles. Whether such aperfluorocarbon is a gas, liquid, or solid depends, of course, on itsliquid/gas or solid/gas phase transition temperature, or boiling point.For example, one of the more preferred perfluorocarbons isperfluoropentane, which has a liquid/gas phase transition temperature orboiling point of 27° C., which means that it will be a liquid atordinary room temperature, but will become a gas in the environment ofthe human body, where the temperature will be above its liquid/gas phasetransition temperature or boiling point. Thus, under normalcircumstance, perfluoropentane is a gaseous precursor. As furtherexamples, there is perfluorobutane and perfluorohexane, the next closesthomologs of perfluoropentane. The liquid/gas phase transitiontemperature of perfluorobutane is 4° C. and that of perfluorohexane is57° C., making the former potentially a gaseous precursor, but generallymore useful as a gas, while the latter would generally be a gaseousprecursor, except under unusual circumstances, because of its highboiling point.

[0069] Another aspect of the present invention is the use of afluorinated compound, especially a perfluorocarbon compound, which willbe in the liquid state at the temperature of use of the vesicles of thepresent invention, to assist or enhance the stability of said gas andgaseous precursor filled vesicles. Such fluorinated compounds includevarious liquid fluorinated compounds, such as fluorinated surfactantsmanufactured by the DuPont Company (Wilmington, Del.), e.g., ZONYL™, aswell as liquid perfluorocarbons. Preferably the fluorinated compoundsare perfluorocarbons. Suitable perfluorocarbons useful as additionalstabilizing agents include perfluorooctylbromide (PFOB),perfluorodecalin, perfluorododecalin, perfluorooctyliodide,perfluorotripropylamine, and perfluorotributylamine. In general,perfluorocarbons over six carbon atoms in length will not be gaseous,i.e., in the gas state, but rather will be liquids, i.e., in the liquidstate, at normal human body temperature. These compounds may, however,additionally be utilized in preparing the stabilized gas and gaseousprecursor filled vesicles used in the present invention. Preferably thisperfluorocarbon is perfluorooctylbromide or perfluorohexane, which is inthe liquid state at room temperature. The gas which is present may be,e.g., nitrogen or perfluoropropane, or may be derived from a gaseousprecursor, which may also be a perfluorocarbon, e.g., perfluoropentane.In that case, the vesicles of the present invention would be preparedfrom a mixture of perfluorocarbons, which for the examples given, wouldbe perfluoropropane (gas) or perfluoropentane (gaseous precursor) andperfluorooctylbromide (liquid). Although not intending to be bound byany theory, it is believed that the liquid fluorinated compound issituated at the interface between the gas and the membrane surface ofthe vesicle. There is thus formed a further stabilizing layer of liquidfluorinated compound on the internal surface of the stabilizingcompound, e.g., a biocompatible lipid used to form the vesicle, and thisperfluorocarbon layer also serves the purpose of preventing the gas fromdiffusing through the vesicle membrane. A gaseous precursor, within thecontext of the present invention, is a liquid or a solid at thetemperature of manufacture and/or storage, but becomes a gas at least ator during the time of use.

[0070] Thus, it has been discovered that a liquid fluorinated compound,such as a perfluorocarbon, when combined with a gas or gaseous precursorordinarily used to make the vesicles of the present invention, mayconfer an added degree of stability not otherwise obtainable with thegas or gaseous precursor alone. Thus, it is within the scope of thepresent invention to utilize a gas or gaseous precursor, such as aperfluorocarbon gaseous precursor, e.g., perfluoropentane, together witha perfluorocarbon which remains liquid after administration to apatient, i.e., whose liquid to gas phase transition temperature is abovethe body temperature of the patient, e.g., perfluoroctylbromide.

[0071] Any biocompatible gas or gaseous precursor may be used to formthe stabilized gas and gaseous precursor filled vesicles. By“biocompatible” is meant a gas or gaseous precursor which, whenintroduced into the tissues of a human patient, will not result in anydegree of unacceptable toxicity, including allergenic responses anddisease states, and preferably are inert. Such a gas or gaseousprecursor should also be suitable for making gas and gaseous precursorfilled vesicles, as described herein.

[0072] The size of the gas or gaseous precursor filled vesicles becomesstabilized when the stabilizing compounds described herein are employed;and the size of the vesicles can then be adjusted for the particularintended MRI end use. For example, magnetic resonance imaging of thevasculature may require vesicles that are no larger that about 30μ indiameter, and that are preferably smaller, e.g., no larger than about12μ in diameter. The size of the gas filled vesicles can be adjusted, ifdesired, by a variety of procedures including microemulsification,vortexing, extrusion, filtration, sonication, homogenization, repeatedfreezing and thawing cycles, extrusion under pressure through pores ofdefined size, and similar methods.

[0073] For intravascular use the vesicles are generally under 30μ inmean diameter, and are preferably under about 12μ in mean diameter. Fortargeted intravascular use, e.g., to bind to a certain tissue such as atumor, the vesicles can be appreciably under a micron, even under 100 nmdiameter. For enteric, i.e., gastrointestinal use the vesicles can bemuch larger, e.g., up to a millimeter in size, but vesicles between 20μand 100μ in mean diameter are preferred.

[0074] As noted above, the embodiments of the present invention may alsoinclude, with respect to their preparation, formation and use, gaseousprecursors that can be activated by temperature. Further below is setout Table I listing a series of gaseous precursors which undergo phasetransitions from liquid to gaseous states at relatively close to normalbody temperature (37° C.) or below, and the size of the emulsifieddroplets that would be required to form a microbubble of a maximum sizeof 10 microns. TABLE I Physical Characteristics of Gaseous Precursorsand Diameter of Emulsified Droplet to Form a 10 μm Vesicle* Diameter(μm) of emulsified droplet Molecular Boiling Point to make 10 micronCompound Weight (° C.) Density vesicle perfluoro 288.04 28.5 1.7326 2.9pentane 1- 76.11 32.5 6.7789 1.2 fluorobutane 2-methyl 72.15 27.8 0.62012.6 butane (isopentane) 2-methyl 1- 70.13 31.2 0.6504 2.5 butene2-methyl-2- 70.13 38.6 0.6623 2.5 butene 1-butene-3- 66.10 34.0 0.68012.4 yne-2-methyl 3-methyl-1- 68.12 29.5 0.6660 2.5 butyne octafluoro200.04 −5.8 1.48 2.8 cyclobutane decafluoro 238.04 −2 1.517 3.0 butanehexafluoro 138.01 −78.1 1.607 2.7 ethane

[0075] There is also set out below a list composed of potential gaseousprecursors that may be used to form vesicles of defined size. However,the list is not intended to be limiting, since it is possible to useother gaseous precursors for that purpose. In fact, for a variety ofdifferent applications, virtually any liquid can be used to make gaseousprecursors so long as it is capable of undergoing a phase transition tothe gas phase upon passing through the appropriate temperature, so thatat least at some point in use it provides a gas. Suitable gaseousprecursors for use in the present invention are the following:hexafluoro acetone, isopropyl acetylene, allene, tetrafluoro-allene,boron trifluoride, isobutane, 1,2-butadiene, 2,3-butadiene,1,3-butadiene, 1,2,3-trichloro-2-fluoro-1,3-butadiene,2-methyl-1,3-butadiene, hexafluoro-1,3-butadiene, butadiyne,1-fluoro-butane, 2-methyl-butane, decafluorobutane, 1-butene, 2-butene,2-methyl-1-butene, 3-methyl-1-butene, perfluoro-1-butene,perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne,butyl nitrate, 1-butyne, 2-butyne,2-chloro-1,1,1,4,4,4-hexafluoro-butyne, 3-methyl-1-butyne,perfluoro-2-butyne, 2-bromo-butyraldehyde, carbonyl sulfide,crotononitrile, cyclobutane, methyl-cyclobutane, octafluoro-cyclobutane,perfluoro-cyclobutene, 3-chlorocyclopentene, octafluorocyclopentene,cyclopropane, 1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane,1,2-dimethyl-cyclopropane, ethylcyclopropane, methylcyclopropane,diacetylene, 3-ethyl-3-methyl diaziridine, 1,1,1-trifluorodiazoethane,dimethyl amine, hexafluorodimethylamine, dimethylethylamine,bis-(dimethylphosphine)amine, perfluorohexane, 2,3-dimethyl-2-norbomane,perfluorodimethylamine, dimethyloxonium chloride, 1,3-dioxolane-2-one,4-methyl-1,1,1,2-tetrafluoroethane, 1,1,1-trifluoroethane,1,1,2,2-tetrafluoroethane, 1,1,2-trichloro-1,2,2-trifluoroethane,1,1-dichloroethane, 1,1-dichloro-1,2,2,2-tetrafluoroethane,1,2-difluoroethane, 1-chloro-1,1,2,2,2-pentafluoroethane,2-chloro-1,1-difluoroethane, 1,1-dichloro-2-fluoroethane,1-chloro-1,1,2,2-tetrafluoroethane, 2-chloro-1,1-difluoroethane,chloroethane, chloropentafluoroethane, dichlorotrifluoroethane,fluoroethane, hexafluoroethane, nitropentafluoroethane,nitrosopentafluoroethane, perfluoroethylamine, ethyl vinyl ether,1,1-dichloroethane, 1,1-dichloro-1,2-difluoroethane, 1,2-difluoroethane,methane, trifluoromethanesulfonylchloride,trifluoromethanesulfonylfluoride, bromodifluoronitrosomethane,bromofluoromethane, bromochlorofluoromethane, bromotrifluoromethane,chlorodifluoronitromethane, chlorodinitromethane, chlorofluoromethane,chlorotrifluoromethane, chlorodifluoromethane, dibromodifluoromethane,dichlorodifluoromethane, dichlorofluoromethane, difluoromethane,difluoroiodomethane, disilanomethane, fluoromethane, iodomethane,iodotrifluoromethane, nitrotrifluoromethane, nitrosotrifluoromethane,tetrafluoromethane, trichlorofluoromethane, trifluoromethane,2-methylbutane, methyl ether, methyl isopropyl ether, methyllactate,methylnitrite, methylsulfide, methyl vinyl ether, neon, neopentane,nitrogen (N₂), nitrous oxide, 1,2,3-nonadecane-tricarboxylicacid-2-hydroxytrimethylester, 1-nonene-3-yne, oxygen (O₂),1,4-pentadiene, n-pentane, perfluoropentane,4-amino-4-methylpentan-2-one, 1-pentene, 2-pentene (cis), 2-pentene(trans), 3-bromopent-1-ene, perfluoropent-1-ene, tetrachlorophthalicacid, 2,3,6-trimethylpiperidine, propane, 1,1,1,2,2,3-hexafluoropropane,1,2-epoxypropane, 2,2-difluoropropane, 2-aminopropane, 2-chloropropane,heptafluoro-1-nitropropane, heptafluoro-1-nitrosopropane,perfluoropropane, propene, hexafluoropropane, 1,1,1,2,3,3-hexafluoro-2,3dichloropropane, 1-chloropropane, chloropropane-(trans),2-chloropropane, 3-fluoropropane, propyne, 3,3,3-trifluoropropyne,3-fluorostyrene, sulfur hexafluoride, sulfur (di)-decafluoride(S₂F₁O),2,4-diaminotoluene, trifluoroacetonitrile, trifluoromethyl peroxide,trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene, vinylether, and xenon.

[0076] The perfluorocarbons, as already indicated, are preferred for useas the gas or gaseous precursors, as well as additional stabilizingcomponents. Included in such perfluorocarbon compositions are saturatedperfluorocarbons, unsaturated perfluorocarbons, and cyclicperfluorocarbons. The saturated perfluorocarbons, which are usuallypreferred, have the formula C_(n)F₂₊₂, where n is from 1 to 12,preferably 2 to 10, more preferably 4 to 8, and most preferably 5.Examples of suitable saturated perfluorocarbons are the following:tetrafluoromethane, hexafluoroethane, octafluoropropane,decafluorobutane, dodecafluoropentane, perfluorohexane, andperfluoroheptane. Cyclic perfluorocarbons, which have the formulaC_(n)F_(2n), where n is from 3 to 8, preferably 3 to 6, may also bepreferred, and include, e.g., hexafluorocyclopropane,octafluorocyclobutane, and decafluorocyclopentane.

[0077] It is part of the present invention to optimize the utility ofthe vesicles by using gases of limited solubility. By limitedsolubility, is meant the ability of the gas to diffuse out of thevesicles by virtue of its solubility in the surrounding aqueous medium.A greater solubility in the aqueous medium imposes a gradient with thegas in the vesicle such that the gas will have a tendency to diffuse outof said vesicle. A lesser solubility in the aqueous milieu, will, on theother hand, decrease or eliminate the gradient between the vesicle andthe interface such that the diffusion of the gas out of the vesicle willbe impeded. Preferably, the gas entrapped in the vesicle has asolubility less than that of oxygen, i.e., 1 part gas in 32 parts water.See Matheson Gas Data Book, 1966, Matheson Company Inc. More preferably,the gas entrapped in the vesicle possesses a solubility in water lessthan that of air; and even more preferably, the gas entrapped in thevesicle possesses a solubility in water less than that of nitrogen.

[0078] Stabilizing Compounds

[0079] One or more stabilizing compounds are employed to form thevesicles, and to assure continued encapsulation of the gases or gaseousprecursors. Even for relatively insoluble, non-diffusible gases such asperfluoropropane or sulfur hexafluoride, improved vesicle preparationsare obtained when one or more stabilizing compounds are utilized in theformation of the gas and gaseous precursor filled vesicles. Thesecompounds maintain the stability and the integrity of the vesicles withregard to their size, shape and/or other attributes.

[0080] The terms “stable” or “stabilized”, as used herein, means thatthe vesicles are substantially resistant to degradation, i.e., areresistant to the loss of vesicle structure or encapsulated gas orgaseous precursor for a useful period of time. Typically, the vesiclesof the invention have a good shelf life, often retaining at least about90 percent by volume of its original structure for a period of at leastabout two or three weeks under normal ambient conditions, although it ispreferred that this period be at least a month, more at least preferablytwo months, even more preferably at least six months, still morepreferably eighteen months, and most preferably three years. Thus, thegas and gaseous precursor filled vesicles typically have a good shelflife, sometimes even under adverse conditions, such as temperatures andpressures which are above or below those experienced under normalambient conditions.

[0081] The stability of the vesicles of the present invention isattributable, at least in part, to the materials from which saidvesicles are made, and it is often not necessary to employ additionalstabilizing additives, although it is optional and often preferred to doso; and such additional stabilizing agents and their characteristics areexplained in more detail herein. The materials from which the vesiclesused in the present invention are constructed are preferablybiocompatible lipid or polymer materials, and of these, thebiocompatible lipids are especially preferred. In addition, because ofthe ease of formulation, i.e., the ability to produce the vesicles justprior to administration, these vesicles may be conveniently made onsite.

[0082] The lipids and polymers employed in preparing the vesicles of theinvention are biocompatible. By “biocompatible” is meant a lipid orpolymer which, when introduced into the tissues of a human patient, willnot result in any degree of unacceptable toxicity, including allergenicresponses and disease states. Preferably the lipids or polymers areinert.

[0083] Biocompatible Lipids

[0084] For the biocompatible lipid materials, it is preferred that suchlipid materials be what is often referred to as “amphiphilic” in nature(i.e., polar lipid), by which is meant any composition of matter whichhas, on the one hand, lipophilic, i.e., hydrophobic properties, while onthe other hand, and at the same time, having lipophobic, i.e.,hydrophilic properties.

[0085] Hydrophilic groups may be charged moieties or other groups havingan affinity for water. Natural and synthetic phospholipids are examplesof lipids useful in preparing the stabilized vesicles used in thepresent invention. They contain charged phosphate “head” groups whichare hydrophilic, attached to long hydrocarbon tails, which arehydrophobic. This structure allows the phospholipids to achieve a singlebilayer (unilamellar) arrangement in which all of the water-insolublehydrocarbon tails are in contact with one another, leaving the highlycharged phosphate head regions free to interact with a polar aqueousenvironment. It will be appreciated that a series of concentric bilayersare possible, i.e., oligolamellar and multilamellar, and sucharrangements are also contemplated to be an aspect of the presentinvention. The ability to form such bilayer arrangements is one featureof the lipid materials useful in the present invention.

[0086] The lipid may alternatively be in the form of a monolayer, andthe monolayer lipids may be used to form a single monolayer(unilamellar) arrangement. Alternatively, the monolayer lipid may beused to form a series of concentric monolayers, i.e., oligolamellar ormultilamellar, and such arrangements are also considered to be withinthe scope of the invention.

[0087] It has also been found advantageous to achieving the stabilizedvesicles of the present invention that they be prepared at a temperaturebelow the gel to liquid crystalline phase transition temperature of alipid used as the stabilizing compound. This phase transitiontemperature is the temperature at which a lipid bilayer will convertfrom a gel state to a liquid crystalline state. See, for example,Chapman et al., J. Biol. Chem. 1974 249, 2512-2521.

[0088] It is believed that, generally, the higher the gel state toliquid crystalline state phase transition temperature, the moreimpermeable the gas and gaseous precursor filled vesicles are at anygiven temperature. See Derek Marsh, CRC Handbook of Lipid Bilayers (CRCPress, Boca Raton, Fla. 1990), at p. 139 for main chain meltingtransitions of saturated diacyl-sn-glycero-3-phosphocholines. The gelstate to liquid crystalline state phase transition temperatures ofvarious lipids will be readily apparent to those skilled in the art andare described, for example, in Gregoriadis, ed., Liposome Technology,Vol. I, 1-18 (CRC Press, 1984). Table 2, below, lists some of therepresentative lipids and their phase transition temperatures: TABLE 2Saturated Diacyl sn-Glycero(3)Phosphocholines: Main Chain PhaseTransition Temperatures* Carbons in Acyl Main Phase Transition ChainsTemperature ° C. 1,2-(12:0) −1.0 1,2-(13:0) 13.7 1,2-(14:0) 23.51,2-(15:0) 34.5 1,2-(16:0) 41.4 1,2-(17:0) 48.2 1,2-(18:0) 55.11,2-(19:0) 61.8 1,2-(20:0) 64.5 1,2-(21:0) 71.1 1,2-(22:0) 74.01,2-(23:0) 79.5 1,2-(24:0) 80.1

[0089] It has been found possible to enhance the stability of thevesicles used in the present invention by incorporating at least a smallamount, i.e., about 1 to about 10 mole percent of the total lipid, of anegatively charged lipid into the lipid from which the gas and gaseousprecursor filled vesicles are to be formed. Suitable negatively chargedlipids include, e.g., phosphatidylserine, phosphatidic acid, and fattyacids. Such negatively charged lipids provide added stability bycounteracting the tendency of the vesicles to rupture by fusingtogether, i.e., the negatively charged lipids tend to establish auniform negatively charged layer on the outer surface of the vesicle,which will be repulsed by a similarly charged outer layer on the othervesicles. In this way, the vesicles will tend to be prevented fromcoming into touching proximity with each other, which would often leadto a rupture of the membrane or skin of the respective vesicles andconsolidation of the contacting vesicles into a single, larger vesicle.A continuation of this process of consolidation will, of course, lead tosignificant degradation of the vesicles.

[0090] The lipid material or other stabilizing compound used to form thevesicles is also preferably flexible, by which is meant, in the contextof gas and gaseous precursor filled vesicles, the ability of a structureto alter its shape, for example, in order to pass through an openinghaving a size smaller than the vesicle.

[0091] In selecting a lipid for preparing the stabilized vesicles usedin the present invention, a wide variety of lipids will be found to besuitable for their construction. Particularly useful are any of thematerials or combinations thereof known to those skilled in the art assuitable for liposome preparation. The lipids used may be of eithernatural, synthetic, or semi-synthetic origin.

[0092] Lipids which may be used to prepare the gas and gaseous precursorfilled vesicles used in the present invention include but are notlimited to: lipids such as fatty acids, lysolipids, phosphatidylcholinewith both saturated and unsaturated lipids includingdioleoylphosphatidylcholine; dimyristoylphosphatidylcholine;dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine;dipalmitoylphosphatidylcholine (DPPC); distearoyl-phosphatidylcholine(DSPC); phosphatidylethanolamines such asdioleoylphosphatidylethanolamine anddipalmitoyl-phosphatidylethanolamine (DPPE); phosphatidylserine;phosphatidylglycerol; phosphatidylinositol; sphingolipids such assphingomyelin; glycolipids such as ganglioside GM1 and GM2; glucolipids;sulfatides; glycosphingolipids; phosphatidic acids such asdipalymitoylphosphatidic acid (DPPA); paltnitic acid; stearic acid;arachidonic acid; oleic acid; lipids bearing polymers such aspolyethylene glycol, i.e., PEGylated lipids, chitin, hyaluronic acid orpolyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, oligo- orpolysaccharides; cholesterol, cholesterol sulfate and cholesterolhemisuccinate; tocopherol hemisuccinate; lipids with ether andester-linked fatty acids; polymerized lipids (a wide variety of whichare well known in the art); diacetyl phosphate; dicetyl phosphate;stearylamine; cardiolipin; phospholipids with short chain fatty acids of6-8 carbons in length; synthetic phospholipids with asymmetric acylchains (e.g., with one acyl chain of 6 carbons and another acyl chain of12 carbons); ceramides; non-ionic liposomes including niosomes such aspolyoxyethylene fatty acid esters, polyoxyethylene fatty alcohols,polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fattyacid esters, glycerol polyethylene glycol oxystearate, glycerolpolyethylene glycol ricinoleate, ethoxylated soybean sterols,ethoxylated castor oil, polyoxyethylene-polyoxypropylene polymers, andpolyoxyethylene fatty acid stearates; sterol aliphatic acid estersincluding cholesterol sulfate, cholesterol butyrate, cholesteroliso-butyrate, cholesterol palmitate, cholesterol stearate, Ianosterolacetate, ergosterol palmitate, and phytosterol n-butyrate; sterol estersof sugar acids including cholesterol glucuroneide, Ianosterolglucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide,cholesterol gluconate, Ianosterol gluconate, and ergosterol gluconate;esters of sugar acids and alcohols including lauryl glucuronide,stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoylgluconate, and stearoyl gluconate; esters of sugars and aliphatic acidsincluding sucrose laurate, fructose laurate, sucrose palmitate, sucrosestearate, glucuronic acid, gluconic acid, accharic acid, and polyuronicacid; saponins including sarsasapogenin, smilagenin, hederagenin,oleanolic acid, and digitoxigenin; glycerol dilaurate, glyceroltrilaurate, glycerol dipalmitate, glycerol and glycerol esters includingglycerol tripalmitate, glycerol distearate, glycerol tristearate,glycerol dimyristate, glycerol trimyristate; longchain alcoholsincluding n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetylalcohol, and n-octadecyl alcohol;6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-α-D-galactopyranoside;6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside;12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoicacid; N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoyl]-2-aminopalmitic acid;cholesteryl)4′-trimethyl-ammonio)butanoate;N-succinyldioleoylphosphatidylethanol-amine; 1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoyl-glycerophosphoethanolamineand palmitoylhomocysteine, and/or combinations thereof.

[0093] If desired, a variety of cationic lipids such as DOTMA,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride; DOTAP,1,2-dioleoyloxy-3-(trimethylammonio)propane; and DOTB,1,2-dioleoyl-3-(4′-trimethyl-ammonio)butanoyl-sn-glycerol may be used.In general the molar ratio of cationic lipid to non-cationic lipid inthe liposome may be, for example, 1:1000, 1:100, preferably, between 2:1to 1:10, more preferably in the range between 1:1 to 1:2.5 and mostpreferably 1:1 (ratio of mole amount cationic lipid to mole amountnon-cationic lipid, e.g., DPPC). A wide variety of lipids may comprisethe non-cationic lipid when cationic lipid is used to construct thevesicle. Preferably, this non-cationic lipid isdipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine ordioleoylphosphatidyl-ethanolamine. In lieu of cationic lipids asdescribed above, lipids bearing cationic polymers such as polylysine orpolyarginine, as well as alkyl phosphonates, alkyl phosphinates, andalkyl phosphites, may also be used to construct the vesicles.

[0094] The most preferred lipids are phospholipids, preferably DPPC,DPPE, DPPA and DSPC, and most preferably DPPC.

[0095] In addition, examples of saturated and unsaturated fatty acidsthat may be used to prepare the stabilized vesicles used in the presentinvention, in the form of gas and gaseous precursor filled mixedmicelles, may include molecules that may contain preferably between 12carbon atoms and 22 carbon atoms in either linear or branched form.Hydrocarbon groups consisting of isoprenoid units and/or prenyl groupscan be used as well. Examples of saturated fatty acids that are suitableinclude, but are not limited to, lauric, myristic, palmitic, and stearicacids; examples of unsaturated fatty acids that may be used are, but arenot limited to, lauroleic, physeteric, myristoleic, palmitoleic,petroselinic, and oleic acids; examples of branched fatty acids that maybe used are, but are not limited to, isolauric, isomyristic,isopalmitic, and isostearic acids. In addition, to the saturated andunsaturated groups, gas and gaseous precursor filled mixed micelles canalso be composed of 5 carbon isoprenoid and prenyl groups. In addition,partially fluorinated phospholipids can be used as stabilizing compoundsfor coating the vesicles.

[0096] Biocompatible Polymers

[0097] The biocompatible polymers useful as stabilizing compounds forpreparing the gas and gaseous precursor filled vesicles used in thepresent invention can be of either natural, semi-synthetic (modifiednatural) or synthetic origin. As used herein, the term polymer denotes acompound comprised of two or more repeating monomeric units, andpreferably 10 or more repeating monomeric units. The phrasesemi-synthetic polymer (or modified natural polymer), as employedherein, denotes a natural polymer that has been chemically modified insome fashion. Exemplary natural polymers suitable for use in the presentinvention include naturally occurring polysaccharides. Suchpolysaccharides include, for example, arabinans, fructans, fucans,galactans, galacturonans, glucans, mannans, xylans (such as, forexample, inulin), levan, fucoidan, carrageenan, galatocarolose, pecticacid, pectin, amylose, pullulan, glycogen, amylopectin, cellulose,dextran, pustulan, chitin, agarose, keratan, chondroitan dermatan,hyaluronic acid, alginic acid, xanthan gum, starch and various othernatural homopolymer or heteropolymers such as those containing one ormore of the following aldoses, ketoses, acids or amines: erythrose,threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose,mannose, gulose, idose, galactose, talose, erythrulose, ribulose,xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol,lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine,threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid,glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconicacid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine,galactosamine, and neuraminic acid, and naturally occurring derivativesthereof. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers suitable for use in the present inventioninclude polyethylenes (such as, for example, polyethylene glycol,polyoxyethylene, and polyethylene terephthlate), polypropylenes (suchas, for example, polypropylene glycol), polyurethanes (such as, forexample, polyvinyl alcohol (PVA), polyvinylchloride andpolyvinylpyrrolidone), polyamides including nylon, polystyrene,polylactic acids, fluorinated hydrocarbons, fluorinated carbons (suchas, for example, polytetrafluoroethylene), and polymethylmethacrylate,and derivatives thereof. Methods for the preparation of suchpolymer-based vesicles will be readily apparent to those skilled in theart, once armed with the present disclosure, when the present disclosureis coupled with information known in the art, such as that described andreferred to in Unger, U.S. Pat. No. 5,205,290, the disclosures of whichare hereby incorporated herein by reference, in their entirety.

[0098] Preferably, when intended to be used in the gastrointestinaltract, the polymer employed is one which has a relatively high waterbinding capacity. When used, for example, in the gastrointestinalregion, a polymer with a high water binding capacity binds a largeamount of free water, enabling the polymer to carry a large volume ofliquid through the gastrointestinal tract, thereby filling anddistending the tract. The filled and distended gastrointestinal tractpermits a clearer picture of the region. In addition, where imaging ofthe gastrointestinal region is desired, preferably the polymer employedis also one which is not substantially degraded within and absorbed fromthe gastrointestinal region. Minimization of metabolism and absorptionwithin the gastrointestinal tract is preferable, so as to avoid theremoval of the contrast agent from the tract as well as avoid theformation of gas within the tract as a result of this degradation.Moreover, particularly where gastrointestinal usage is contemplated,polymers are preferably such that they are capable of displacing air andminimizing the formation of large air bubbles within the polymercomposition.

[0099] Particularly preferred embodiments of the present inventioninclude vesicles wherein the stabilizing compound from which thestabilized gas and gaseous precursor filled vesicles are formedcomprises three components: (1) a neutral (e.g., nonionic orzwitterionic) lipid, (2) a negatively charged lipid, and (3) a lipidbearing a hydrophilic polymer. Preferably, the amount of said negativelycharged lipid will be greater than 1 mole percent of total lipidpresent, and the amount of lipid bearing a hydrophilic polymer will begreater than 1 mole percent of total lipid present. It is also preferredthat said negatively charged lipid be a phosphatidic acid. The lipidbearing a hydrophilic polymer will desirably be a lipid covalently boundto said polymer, and said polymer will preferably have a weight averagemolecular weight of from about 400 to about 100,000. Said hydrophilicpolymer is preferably selected from the group consisting ofpolyethyleneglycol, polypropyleneglycol, polyvinylalcohol, andpolyvinylpyrrolidone and copolymers thereof. The PEG or other polymermay be bound to the DPPE or other lipid through a covalent linkage, suchas through an amide, carbamate or amine linkage. Alternatively, ester,ether, thioester, thioamide or disulfide (thioester) linkages may beused with the PEG or other polymer to bind the polymer to, for example,cholesterol or other phospholipids. Where the hydrophilic polymer ispolyethyleneglycol, a lipid bearing such a polymer will be said to be“PEGylated,” which has reference to the abbreviation forpolyethyleneglycol: “PEG.” Said lipid bearing a hydrophilic polymer ispreferably dipalmitoylphosphatidylethanolamine-polyethyleneglycol 5000,i.e., a dipalmitoylphosphatidylethanolamine lipid having apolyethyleneglycol polymer of a mean weight average molecular weight ofabout 5000 attached thereto (DPPE-PEG5000); ordistearoyl-phosphatidylethanolamine-polyethyleneglycol 5000.

[0100] Preferred embodiments of the vesicle contemplated by the presentinvention would include, e.g., 77.5 mole percentdipalmitoylphophatidylcholine (DPPC), with 12.5 mole percent ofdipalmitoylphosphatidic acid (DPPA), and with 10 mole percent ofdipalmitoylphosphatidylethanolamine-polyethyleneglycol-5000(DPPE/PEG5000). These compositions in a 82/10/8 ratio of molepercentages, respectively, is also preferred. The DPPC component iseffectively neutral, since the phosphtidyl portion is negatively chargedand the choline portion is positively charged. Consequently, the DPPAcomponent, which is negatively charged, is added to enhancestabilization in accordance with the mechanism described further aboveregarding negatively charged lipids as an additional agent. The thirdcomponent, DPPE/PEG, provides a PEGylated material bound to the lipidmembrane or skin of the vesicle by the DPPE moiety, with the PEG moietyfree to surround the vesicle membrane or skin, and thereby form aphysical barrier to various enzymatic and other endogenous agents in thebody whose function is to degrade such foreign materials. It is alsotheorized that the PEGylated material, because of its structuralsimilarity to water, is able to defeat the action of the macrophages ofthe human immune system, which would otherwise tend to surround andremove the foreign object. The result is an increase in the time duringwhich the stabilized vesicles can function as contrast media.

[0101] Other and Auxiliary Stabilizing Compounds

[0102] It is also contemplated to be a part of the present invention toprepare stabilized gas and gaseous precursor filled vesicles usingcompositions of matter in addition to the biocompatible lipids andpolymers described above, provided that the vesicles so prepared meetthe stability and other criteria set forth herein. These compositionsmay be basic and fundamental, i.e., form the primary basis for creatingor establishing the stabilized gas and gaseous precursor filledvesicles. On the other hand, they may be auxiliary, i.e., act assubsidiary or supplementary agents which either enhance the functioningof the basic stabilizing compound or compounds, or else contribute somedesired property in addition to that afforded by the basic stabilizingcompound.

[0103] However, it is not always possible to determine whether a givencompound is a basic or an auxiliary agent, since the functioning of thecompound in question is determined empirically, i.e., by the resultsproduced with respect to producing stabilized vesicles. As examples ofhow these basic and auxiliary compounds may function, it has beenobserved that the simple combination of a biocompatible lipid and wateror saline when shaken will often give a cloudy solution subsequent toautoclaving for sterilization. Such a cloudy solution may function as acontrast agent, but is aesthetically objectionable and may implyinstability in the form of undissolved or undispersed lipid particles.Thus, propylene glycol may be added to remove this cloudiness byfacilitating dispersion or dissolution of the lipid particles. Thepropylene glycol may also function as a thickening agent which improvesvesicle formation and stabilization by increasing the surface tension onthe vesicle membrane or skin. It is possible that the propylene glycolfurther functions as an additional layer that coats the membrane or skinof the vesicle, thus providing additional stabilization. As examples ofsuch further basic or auxiliary stabilizing compounds, there areconventional surfactants which may be used; see D'Arrigo U.S. Pat. Nos.4,684,479 and 5,215,680.

[0104] Additional auxiliary and basic stabilizing compounds include suchagents as peanut oil, canola oil, olive oil, safflower oil, corn oil, orany other oil commonly known to be ingestible which is suitable for useas a stabilizing compound in accordance with the requirements andinstructions set forth in the instant specification.

[0105] In addition, compounds used to make mixed micelle systems may besuitable for use as basic or auxiliary stabilizing compounds, and theseinclude, but are not limited to: lauryltrimethylammonium bromide(dodecyl-), cetyltrimethylammonium bromide (hexadecyl-),myristyltrimethylammonium bromide (tetradecyl-),alkyldimethylbenzylammonium chloride (alkyl=C₁₂, C₁₄, C₁₆,),benzyldimethyldodecylammonium bromide/chloride, benzyldimethylhexadecylammonium bromide/chloride, benzyldimethyl tetradecylammoniumbromide/chloride, cetyldimethylethylammonium bromide/chloride, orcetylpyridinium bromide/chloride.

[0106] It has been found that the gas and gaseous precursor filledvesicles used in the present invention may be controlled according tosize, solubility and heat stability by choosing from among the variousadditional or auxiliary stabilizing agents described herein. Theseagents can affect theseparameters of the vesicles not only by theirphysical interaction with the lipid coatings, but also by their abilityto modify the viscosity and surface tension of the surface of the gasand gaseous precursor filled vesicle. Accordingly, the gas and gaseousprecursor filled vesicles used in the present invention may be favorablymodified and further stabilized, for example, by the addition of one ormore of a wide variety of (a) viscosity modifiers, including, but notlimited to carbohydrates and their phosphorylated and sulfonatedderivatives; and polyethers, preferably with molecular weight rangesbetween 400 and 100,000; di- and trihydroxy alkanes and their polymers,preferably with molecular weight ranges between 200 and 50,000; (b)emulsifying and/or solubilizing agents may also be used in conjunctionwith the lipids to achieve desired modifications and furtherstabilization; such agents include, but are not limited to, acacia,cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohols,lecithin, mono- and di-glycerides, mono-ethanolamine, oleic acid, oleylalcohol, poloxamer (e.g., poloxamer 188, poloxamer 184, and poloxamer181), polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate,polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,propylene glycol diacetate, propylene glycol monostearate, sodium laurylsulfate, sodium stearate, sorbitan mono-laurate, sorbitan mono-oleate,sorbitan mono-palmitate, sorbitan monostearate, stearic acid, trolamine,and emulsifying wax; (c) suspending and/or viscosity-increasing agentsthat may be used with the lipids include, but are not limited to,acacia, agar, alginic acid, aluminum mono-stearate, bentonite, magma,carbomer 934P, carboxymethylcellulose, calcium and sodium and sodium 12,carrageenan, cellulose, dextran, gelatin, guar gum, locust bean gum,veegum, hydroxyethyl cellulose, hydroxypropyl methylcellulose,magnesium-aluminum-silicate, methylcellulose, pectin, polyethyleneoxide, povidone, propylene glycol alginate, silicon dioxide, sodiumalginate, tragacanth, xanthum gum, α-d-gluconolactone, glycerol andmannitol; (d) synthetic suspending agents may also be utilized such aspolyethyleneglycol (PEG), polyvinylpyrrolidone (PVP), polyvinylalcohol(PVA), polypropylene glycol, and polysorbate; and (e) tonicity raisingagents may be included; such agents include but are not limited tosorbitol, propyleneglycol and glycerol.

[0107] Aqueous Diluents

[0108] As mentioned earlier, where the vesicles are lipid in nature, aparticularly desired component of the stabilized vesicles is an aqueousenvironment of some kind, which induces the lipid, because of itshydrophobic/hydrophilic nature, to form vesicles, the most stableconfiguration which it can achieve in such an environment. The diluentswhich can be employed to create such an aqueous environment include, butare not limited to water, either deionized or containing any number ofdissolved salts, etc., which will not interfere with creation andmaintenance of the stabilized vesicles or their use as MRI contrastagents; and normal saline and physiological saline.

[0109] Paramagnetic and Superparamagnetic Contrast Agents

[0110] In a further embodiment of the present invention, the stabilizedgas filled vesicle based contrast medium of the invention may furthercomprise additional contrast agents such as conventional contrastagents, which may serve to increase the efficacy of the contrast mediumfor simultaneous magnetic resonance focused noninvasive ultrasound. Manysuch contrast agents are well known to those skilled in the art andinclude paramagnetic and superparamagnetic contrast agents.

[0111] Exemplary paramagnetic contrast agents suitable for use in thesubject invention include stable free radicals (such as, for example,stable nitroxides), as well as compounds comprising transition,lanthanide and actinide elements, which may, if desired, be in the formof a salt or may be covalently or noncovalently bound to complexingagents (including lipophilic derivatives thereof) or to proteinaceousmacromolecules.

[0112] Preferable transition, lanthanide and actinide elements includeGd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II),Ni(II), Eu(III) and Dy(III). More preferably, the elements includeGd(III), Mn(II), Cu(II), Fe(II), Fe(III), Eu(III) and Dy(III),especially Mn(II) and Gd(III).

[0113] These elements may, if desired, be in the form of a salt, such asa manganese salt, e.g., manganese chloride, manganese carbonate,manganese acetate, and organic salts of manganese such as manganesegluconate and manganese hydroxylapatite; and such as an iron salt, e.g.,iron sulfides and ferric salts such as ferric chloride.

[0114] These elements may also, if desired, be bound, e.g., covalentlyor noncovalently, to complexing agents (including lipophilic derivativesthereof) or to proteinaceous macromolecules. Preferable complexingagents include, for example, diethylenetriamine-pentaacetic acid (DTPA),ethylene-diaminetetraacetic acid (EDTA),1,4,7,10-tetraazacyclododecane-N,N′,N′,N′″-tetraacetic acid (DOTA),1,4,7,10-tetraazacyclododecane-N,N′,N″-triacetic acid (DO3A),3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-tridecanoicacid (B-19036), hydroxybenzylethylene-diamine diacetic acid (HBED),N,N′-bis(pyridoxyl-5-phosphate)ethylene diamine, N,N′-diacetate (DPDP),1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA),1,4,8,11-tetraazacyclotetradecane-N,N′N″,N′″-tetraacetic acid (TETA),kryptands (that is, macrocyclic complexes), and desferrioxamine. Morepreferably, the complexing agents are EDTA, DTPA, DOTA, DO3A andkryptands, most preferably DTPA. Preferable lipophilic complexes thereofinclude alkylated derivatives of the complexing agents EDTA, DOTA, etc.,for example, EDTA-DDP, that is,N,N′-bis-(carboxy-decylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate;EDTA-ODP, that isN,N′-bis-(carboxy-octadecylamido-methyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate;EDTA-LDPN,N′-Bis-(carboxy-laurylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate; etc.; such as those described in U.S. Ser. No. 887,290,filed May 22, 1992, the disclosures of which are hereby incorporatedherein by reference in its entirety. Preferable proteinaceousmacromolecules include albumin, collagen, polyarginine, polylysine,polyhistidine, γ-globulin and β-globulin. More preferably, theproteinaceous macromolecules comprise albumin, polyarginine, polylysine,and polyhistidine.

[0115] Suitable complexes thus include Mn(II)-DTPA, Mn(II)-EDTA,Mn(II)-DOTA, Mn(II)-DO3A, Mn(II)-kryptands, Gd(III)-DTPA, Gd(III)-DOTA,Gd(III)-DO3A, Gd(III)-kryptands, Cr(III)-EDTA, Cu(II)-EDTA, oriron-desferrioxamine, especially Mn(II)-DTPA or Gd(III)-DTPA.

[0116] Paramagnetic chelates, such as alkylated chelates of paramagneticions, as disclosed in U.S. Pat. No. 5,312,617, the disclosure of whichis incorporated herein by reference in its entirety, paramagneticcopolymeric chelates as in U.S. Pat. No. 5,385,719 useful for attachingto gas filled liposomes and to the surface of gas filled polymericliposomes, nitroxide stable free radicals (NSFRs) useful for attachingto lipids in gas filled liposomes as well as to polymers forconstruction gas filled liposomes and hybrid complexes comprised ofchelate moieties containing one or more paramagnetic ions in closeproximity with one or more NSFRs as outlined in U.S. Pat. No. 5,407,657,may be used for constructing paramagnetic gas filled liposomes. Thesehybrid complexes have greatly increased relaxivity and thereforeincrease the sensitivity to the vesicle on magnetic resonance.Nitroxides are paramagnetic contrast agents which increase both T1 andT2 relaxation rates by virtue of one unpaired electron in the nitroxidemolecule. The paramagnetic effectiveness of a given compound as an MRIcontrast agent is at least partly related to the number of unpairedelectrons in the paramagnetic nucleus or molecule, specifically to thesquare of the number of unpaired electrons. For example, gadolinium hasseven unpaired electrons and a nitroxide molecule has only one unpairedelectron; thus gadolinium is generally a much stronger MRI contrastagent than a nitroxide. However, effective correlation time, anotherimportant parameter for assessing the effectiveness of contrast agents,confers potential increased relaxivity to the nitroxides. When theeffective correlation time is very close to the proton Larmourfrequency, the relaxation rate may increase dramatically. When thetumbling rate is slowed, e.g., by attaching the paramagnetic contrastagent to a large structure, it will tumble more slowly and thereby moreeffectively transfer energy to hasten relaxation of the water protons.In gadolinium, however, the electron spin relaxation time is rapid andwill limit the extent to which slow rotational correlation times canincrease relaxivity. For nitroxides, however, the electron spincorrelation times are more favorable and tremendous increases inrelaxivity may be attained by slowing the rotational correlation time ofthese molecules. The gas filled vesicles of the present invention areideal for attaining the goals of slowed rotational correlation times andresultant improvement in relaxivity. Although not intending to be boundby any particular theory of operation, it is contemplated that since thenitroxides may be designed to coat the perimeters of the gas filledvesicles, e.g., by making alkyl derivatives thereof, that the resultingcorrelation times can be optimized. Moreover, the resulting contrastmedium of the present invention may be viewed as a magnetic sphere, ageometric configuration which maximizes relaxivity.

[0117] If desired, the nitroxides may be alkylated or otherwisederivitized, such as the nitroxides2,2,5,5-tetramethyl-1-pyrrolidinyloxy, free radical, and2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TMPO).

[0118] Exemplary superparamagnetic contrast agents suitable for use inthe subject invention include metal oxides and sulfides which experiencea magnetic domain, ferro- or ferrimagnetic compounds, such as pure iron,magnetic iron oxide (such as magnetite), γ-Fe₂O₃, Fe₃O₄, iron sulfides,manganese ferrite, cobalt, ferrite, nickel ferrite, and ferritin filledwith magnetite or other magnetically active materials such asferromagnetic and superparamagnetic materials.

[0119] The contrast agents, such as the paramagnetic andsuperparamagnetic contrast agents described above, may be employed as acomponent within the vesicles or in the contrast medium comprising thevesicles. They may be entrapped within the internal space of thevesicles, administered as a solution with the vesicles or incorporatedinto the stabilizing compound forming the vesicle wall.

[0120] Superparamagnetic agents may be used as clathrates to adsorb andstabilize vesicles. For example, emulsions of various perfluorocarbons,such as perfluorohexane or perfluorochlorocarbons mixed with irregularshaped iron oxide compounds. The hydrophobic clefts in the iron oxidescause nano-droplets of the liquid gaseous precursor to adhere to thesurface of the solid material.

[0121] For example, if desired, the paramagnetic or superparamagneticagents may be delivered as alkylated or other derivatives incorporatedinto the stabilizing compound, especially the lipidic walls of thevesicles. In particular, the nitroxides2,2,5,5-tetramethyl-1-pyrrolidinyloxy, free radical and2,2,6,6-tetramethyl-1-piperidinyloxy, free radical, can form adductswith long chain fatty acids at the positions of the ring which are notoccupied by the methyl groups, via a number of different linkages, e.g.,an acetyloxy group. Such adducts are very amenable to incorporation intothe stabilizing compounds, especially those of a lipidic nature, whichform the walls of the vesicles of the present invention.

[0122] Mixtures of any one or more of the paramagnetic agents and/orsuperparamagnetic agents in the contrast media may similarly be used.

[0123] The paramagnetic and superparamagnetic agents described above mayalso be coadministered separately, if desired.

[0124] The gas filled vesicles used in the present invention may notonly serve as effective carriers of the superparamagnetic agents, e.g.,iron oxides, but also appear to magnify the effect of the susceptibilitycontrast-agents. Superparamagnetic contrast agents include metal oxides,particularly iron oxides but including manganese oxides, and as ironoxides, containing varying amounts of manganese, cobalt and nickel whichexperience a magnetic domain. These agents are nano or microparticlesand have very high bulk susceptibilities and transverse relaxationrates. The larger particles, e.g., 100 nm diameter, have much higher R2relaxivities than R1 relaxivities but the smaller particles, e.g., 10 to15 nm diameter have somewhat lower R2 relaxivities, but much morebalanced R1 and R2 values. The smallest particles, e.g., monocrystallineiron oxide particles, 3 to 5 nm in diameter, have lower R2 relaxivities,but probably the most balanced R1 and R2 relaxation rates. Ferritin canalso be formulated to encapsulate a core of very high relaxation ratesuperparamagnetic iron. It has been discovered that stabilized gasfilled vesicles used in the present invention can increase the efficacyand safety of these conventional iron oxide based MRI contrast agents.

[0125] The iron oxides may simply be incorporated into the stabilizingcompounds from which the vesicles are made. Particularly, the ironoxides may be incorporated into the walls of the lipid based vesicles,e.g., adsorbed onto the surfaces of the vesicles, or entrapped withinthe interior of the vesicles as described in U.S. Pat. No. 5,088,499,issued Feb. 18, 1992.

[0126] Although there is no intention to limit the present invention toany particular theory as to its mode of action, it is believed that thevesicles increase the efficacy of the superparamagnetic contrast agentsby several mechanisms. First, it is believed that the vesicles functionso as to increase the apparent magnetic concentration of the iron oxideparticles. Second, it is believed that the vesicles increase theapparent rotational correlation time of the MRI contrast agents, bothparamagnetic and superparamagnetic agents, so that relaxation rates areincreased. Finally, the vesicles appear to operate by way of a novelmechanism which increases the apparent magnetic domain of the contrastmedium and is believed to operate in the manner described immediatelybelow.

[0127] The vesicles may be thought of as flexible spherical domains ofdiffering susceptibility from the suspending medium, i.e., the aqueoussuspension of the contrast medium and the gastrointestinal fluids in thecase of gastrointestinal administration, and blood or other body fluidsin the cases of intravascular injection or injection into another bodyspace. When considering ferrites or iron oxide particles, it should benoted that these agents have a particle size dependent effect oncontrast, i.e., it depends on the particle diameter of the iron oxideparticle. This phenomenon is very common and is often referred to as the“secular” relaxation of the water molecules. Described in more physicalterms, this relaxation mechanism is dependent upon the effective size ofthe molecular complex in which a paramagnetic atom, or paramagneticmolecule, or molecules, may reside. One physical explanation may bedescribed in the following Solomon-Bloembergen equations which definethe paramagnetic contributions to the T₁ and T₂ relaxation times of aspin ½ nucleus with gyromagnetic ratio g perturbed by a paramagneticion:

1/T ₁ M=({fraction (2/15)})S(S+1)γ² g ²β² /r ⁶[3τ_(c)/(1+ω_(I) ²τ_(c)²)+7τ_(c)/(1+ω_(s) ²τ_(c) ²)]+(⅔)S(S+1)A ² /h ²[τ_(e)/(1+ω_(s)2τ_(e) ²)]

[0128] and

1/T ₂ M=({fraction (1/15)})S(S+1)γ² g ²β² /r ⁶[4τ_(c)/(1+ω_(I) ²τ_(c)²)+13τ_(c)/(1+ω_(s) ²τ_(c) ²)]+(⅓)S(S+1)A ² /h ²[τ_(e)/(1+ω_(s)2τ_(e)²)]

[0129] where:

[0130] S=electron spin quantum number;

[0131] g=electronic g factor;

[0132] γ=Bohr magneton;

[0133] ω_(I) and ω_(s) (=657 w_(I))=Larmor angular precessionfrequencies for the nuclear spins and electron spins;

[0134] r=ion-nucleus distance;

[0135] A=hyperfine coupling constant;

[0136] τ_(c) and τ_(e)=correlation times for the dipolar and scalarinteractions, respectively; and

[0137] h=Planck's constant.

[0138] See, e.g., Solomon, I. Phys. Rev. 99, 559 (1955) and Bloembergen,N. J. Chem. Phys. 27, 572, 595 (1957), the disclosures of which arehereby incorporated by reference in their entirety.

[0139] A few large particles will generally have a much greater effectthan a larger number of much smaller particles, primarily due to alarger correlation time. If one were to make the iron oxide particlesvery large however, they might be toxic and embolize the lungs oractivate the complement cascade system. Furthermore, it is not the totalsize of the particle that matters, but particularly the diameter of theparticle at its edge or outer surface. The domain of magnetization orsusceptibility effect falls off exponentially from the surface of theparticle. Generally speaking, in the case of dipolar (through space)relaxation mechanisms, this exponential fall off exhibits an r⁶dependence. Literally interpreted, a water molecule that is 4 angstromsaway from a paramagnetic surface will be influenced 64 times less than awater molecule that is 2 angstroms away from the same paramagneticsurface. The ideal situation in terms of maximizing the contrast effectwould be to make the iron oxide particles hollow, flexible and as largeas possible. Up until now it has not been possible to do this;furthermore, these benefits have probably been unrecognized until now.By coating the inner or outer surfaces of the vesicles with the contrastagents, even though the individual contrast agents, e.g., iron oxidenanoparticles or paramagnetic ions, are relatively small structures, theeffectiveness of the contrast agents may be greatly enhanced. In sodoing, the contrast agents may function as an effectively much largersphere wherein the effective domain of magnetization is determined bythe diameter of the vesicle and is maximal at the surface of thevesicle. These agents afford the advantage of flexibility, i.e.,compliance. While rigid vesicles might lodge in the lungs or otherorgans and cause toxic reactions, these flexible vesicles slide throughthe capillaries much more easily.

[0140] Methods of Preparation

[0141] The stabilized gas filled vesicles used in the present inventionmay be prepared by a number of suitable methods. These are describedbelow separately for the case where the vesicles are gas filled, andwhere they are gaseous precursor filled, although vesicles having both agas and gaseous precursor are part of the present invention.

[0142] Utilizing a Gas

[0143] A preferred embodiment comprises the steps of agitating anaqueous solution comprising a stabilizing compound, preferably a lipid,in the presence of a gas at a temperature below the gel to liquidcrystalline phase transition temperature of the lipid to form gas filledvesicles. The term agitating, and variations thereof, as used herein,means any motion that shakes an aqueous solution such that gas isintroduced from the local ambient environment into the aqueous solution.The shaking must be of sufficient force to result in the formation ofvesicles, particularly stabilized vesicles. The shaking may be byswirling, such as by vortexing, side-to-side, or up-and-down motion.Different types of motion may be combined. Also, the shaking may occurby shaking the container holding the aqueous lipid solution, or byshaking the aqueous solution within the container without shaking thecontainer itself.

[0144] Further, the shaking may occur manually or by machine. Mechanicalshakers that may be used include, for example, a shaker table such as aVWR Scientific (Cerritos, Calif.) shaker table, or a Wig-L-Bug® Shakerfrom Crescent Dental Mfg. Ltd., Lyons, Ill., which has been found togive excellent results. It is a preferred embodiment of the presentinvention that certain modes of shaking or vortexing be used to makestable vesicles within a preferred size range. Shaking is preferred, andit is preferred that this shaking be carried out using the Wig-L-Bug®mechanical shaker. In accordance with this preferred method, it ispreferred that a reciprocating motion be utilized to generate the gasfilled vesicles. It is even more preferred that the motion bereciprocating in the form of an arc. It is still more preferred that themotion be reciprocating in the form of an arc between about 2° and about20°, and yet further preferred that the arc be between about 5° andabout 8°. It is most preferred that the motion is reciprocating betweenabout 6° and about 7°, most particularly about 6.5°. It is contemplatedthat the rate of reciprocation, as well as the arc thereof, is criticalto determining the amount and size of the gas filled vesicles formed. Itis a preferred embodiment of the present invention that the number ofreciprocations, i.e., full cycle oscillations, be within the range ofabout 1000 and about 20,000 per minute. More preferably, the number ofreciprocations or oscillations will be between 2500 and 8000. TheWig-L-Bug®, referred to above, is a mechanical shaker which provides2000 pestle strikes every 10 seconds, i.e., 6000 oscillations everyminute. Of course, the number of oscillations is dependent upon the massof the contents being agitated, with the larger the mass, the fewerthe-number of oscillations). Another means for producing shakingincludes the action of gas emitted under high velocity or pressure.

[0145] It will also be understood that preferably, with a larger volumeof aqueous solution, the total amount of force will be correspondinglyincreased. Vigorous shaking is defined as at least about 60 shakingmotions per minute, and is preferred. Vortexing at least 60-300revolutions per minute is more preferred. Vortexing at 300-1800revolutions per minute is most preferred. The formation of gas filledvesicles upon shaking can be detected visually. The concentration oflipid required to form a desired stabilized vesicle level will varydepending upon the type of lipid used, and may be readily determined byroutine experimentation. For example, in preferred embodiments, theconcentration of 1,2-dipalimitoyl-phosphatidylcholine (DPPC) used toform stabilized vesicles according to the methods of the presentinvention is about 0.1 mg/ml to about 30 mg/ml of saline solution, morepreferably from about 0.5 mg/ml to about 20 mg/ml of saline solution,and most preferably from about 1 mg/ml to about 10 mg/ml of salinesolution. The concentration of distearoylphosphatidylcholine (DSPC) usedin preferred embodiments is about 0.1 mg/ml to about 30 mg/ml of salinesolution, more preferably from about 0.5 mg/ml to about 20 mg/ml ofsaline solution, and most preferably from about 1 mg/ml to about 10mg/ml of saline solution.

[0146] In addition to the simple shaking methods described above, moreelaborate, but for that reason less preferred, methods can also beemployed, e.g., liquid crystalline shaking gas instillation processes,and vacuum drying gas instillation processes, such as those described inU.S. Ser. No. 076,250, filed Jun. 11, 1993, which is incorporated hereinby reference, in its entirety. When such processes are used, thestabilized vesicles which are to be gas filled, may be prepared prior togas installation using any one of a variety of conventional liposomepreparatory techniques which will be apparent to those skilled in theart. These techniques include freeze-thaw, as well as techniques such assonication, chelate dialysis, homogenization, solvent infusion,microemulsification, spontaneous formation, solvent vaporization, Frenchpressure cell technique, controlled detergent dialysis, and others, eachinvolving preparing the vesicles in various fashions in a solutioncontaining the desired active ingredient so that the therapeutic,cosmetic or other agent is encapsulated in, enmeshed in, or attached theresultant polar-lipid based vesicle. See, e.g., Madden et al., Chemistryand Physics of Lipids, 1990 53, 37-46, the disclosure of which is herebyincorporated herein by reference in its entirety.

[0147] The gas filled vesicles prepared in accordance with the methodsdescribed above range in size from below a micron to over 100μ in size.In addition, it will be noted that after the extrusion and sterilizationprocedures, the agitation or shaking step yields gas filled vesicleswith little to no residual anhydrous lipid phase (Bangham, A. D.,Standish, M. M, & Watkins, J. C. (1965) J. Mol. Biol. 13, 238-252)present in the remainder of the solution. The resulting gas filledvesicles remain stable on storage at room temperature for a year or evenlonger.

[0148] The size of gas filled vesicles can be adjusted, if desired, by avariety of procedures including microemulsification, vortexing,extrusion, filtration, sonication, homogenization, repeated freezing andthawing cycles, extrusion under pressure through pores of defined size,and similar methods. It may also be desirable to use the vesicles of thepresent invention as they are formed, without any attempt at furthermodification of the size thereof.

[0149] The gas filled vesicles may be sized by a simple process ofextrusion through filters; the filter pore sizes control the sizedistribution of the resulting gas filled vesicles. By using two or morecascaded, i.e., a stacked set of filters, e.g., 10μ followed by 8μ, thegas filled vesicles have a very narrow size distribution centered around7-9 μm. After filtration, these stabilized gas filled vesicles remainstable for over 24 hours.

[0150] The sizing or filtration step may be accomplished by the use of afilter assembly when the suspension is removed from a sterile vial priorto use, or even more preferably, the filter assembly may be incorporatedinto the syringe itself during use. The method of sizing the vesicleswill then comprise using a syringe comprising a barrel, at least onefilter, and a needle; and will be carried out by a step of extractingwhich comprises extruding said vesicles from said barrel through saidfilter fitted to said syringe between said barrel and said needle,thereby sizing said vesicles before they are administered to a patientin the course of using the vesicles as MRI contrast agents in accordancewith the present invention. The step of extracting may also comprisedrawing said vesicles into said syringe, where the filter will functionin the same way to size the vesicles upon entrance into the syringe.Another alternative is to fill such a syringe with vesicles which havealready been sized by some other means, in which case the filter nowfunctions to ensure that only vesicles within the desired size range, orof the desired maximum size, are subsequently administered by extrusionfrom the syringe.

[0151] Typical of the devices which can be used for carrying out thesizing or filtration step, is the syringe and filter combination shownin FIG. 2 of U.S. application Ser. No. 08/401,974, filed Mar. 9, 1995,the disclosure of which is incorporated by reference in its entirety.

[0152] In preferred embodiments, the stabilizing compound solution orsuspension is extruded through a filter and the said solution orsuspension is heat sterilized prior to shaking. Once gas filled vesiclesare formed, they may be filtered for sizing as described above. Thesesteps prior to the formation of gas filled vesicles provide theadvantages, for example, of reducing the amount of unhydratedstabilizing compound, and thus providing a significantly higher yield ofgas filled vesicles, as well as and providing sterile gas filledvesicles ready for administration to a patient. For example, a mixingvessel such as a vial or syringe may be filled with a filteredstabilizing compound, especially lipid suspension, and the suspensionmay then be sterilized within the mixing vessel, for example, byautoclaving. Gas may be instilled into the lipid suspension to form gasfilled vesicles by shaking the sterile vessel. Preferably, the sterilevessel is equipped with a filter positioned such that the gas filledvesicles pass through the filter before contacting a patient.

[0153] The first step of this preferred method, extruding thestabilizing, especially lipid, solution through a filter, decreases theamount of unhydrated compound by breaking up the dried compound andexposing a greater surface area for hydration. Preferably, the filterhas a pore size of about 0.1 to about 5 μm, more preferably, about 0.1to about 4 μm, even more preferably, about 0.1 to about 2 μm, and mostpreferably, about 1 μm. Unhydrated compound, especially lipid, appearsas amorphous clumps of non-uniform size and is undesirable.

[0154] The second step, sterilization, provides a composition that maybe readily administered to a patient for MRI imaging. Preferably,sterilization is accomplished by heat sterilization, preferably, byautoclaving the solution at a temperature of at least about 100° C., andmore preferably, by autoclaving at about 100° C. to about 130° C., evenmore preferably, about 110° C. to about 130° C., even more preferably,about 120° C. to about 130° C., and most preferably, about 130° C.Preferably, heating occurs for at least about 1 minute, more preferably,about 1 to about 30 minutes, even more preferably, about 10 to about 20minutes, and most preferably, about 15 minutes.

[0155] If desired, alternatively the first and second steps, as outlinedabove, may be reversed, or only one of the two steps employed.

[0156] Where sterilization occurs by a process other than heatsterilization at a temperature which would cause rupture of the gasfilled vesicles, sterilization may occur subsequent to the formation ofthe gas filled vesicles, and is preferred. For example, gamma radiationmay be used before and/or after gas filled vesicles are formed.

[0157] Utilizing a Gaseous Precursor

[0158] In addition to the aforementioned embodiments, one can also usegaseous precursors contained in the lipid-based vesicles that can, uponactivation by temperature, light, or pH, or other properties of thetissues of a host to which it is administered, undergo a phasetransition from a liquid or solid entrapped in the lipid-based vesicles,to a gaseous state, expanding to create the stabilized, gas filledvesicles used in the present invention. This technique is described indetail in patent application Ser. No. 08/160,232, filed Nov. 30, 1993,and Ser. No. 08/159,687, filed Nov. 30, 1993, both of which areincorporated herein by reference in their entirety. The techniques forpreparing gaseous precursor filled vesicles are generally similar tothose described for the preparation of gas filled vesicles herein,except that a gaseous precursor is substituted for the gas.

[0159] The preferred method of activating the gaseous precursor is bytemperature. Activation or transition temperature, and like terms, referto the boiling point of the gaseous precursor, the temperature at whichthe liquid to gaseous phase transition of the gaseous precursor takesplace. Useful gaseous precursors are those gases which have boilingpoints in the range of about −100° C. to 70° C. The activationtemperature is particular to each gaseous precursor. An activationtemperature of about 37° C., or human body temperature, is preferred forgaseous precursors of the present invention. Thus, a liquid gaseousprecursor is activated to become a gas at 37° C. However, the gaseousprecursor may be in liquid or gaseous phase for use in the methods ofthe present invention. The methods of preparing the MRI contrast agentsused in the present invention may be carried out below the boiling pointof the gaseous precursor such that a liquid is incorporated into avesicle. In addition, the said methods may be performed at the boilingpoint of the gaseous precursor such that a gas is incorporated into avesicle. For gaseous precursors having low temperature boiling points,liquid precursors may be emulsified using a microfluidizer devicechilled to a low temperature. The boiling points may also be depressedusing solvents in liquid media to utilize a precursor in liquid form.Further, the methods may be performed where the temperature is increasedthroughout the process, whereby the process starts with a gaseousprecursor as a liquid and ends with a gas.

[0160] The gaseous precursor may be selected so as to form the gas insitu in the targeted tissue or fluid, in vivo upon entering the patientor animal, prior to use, during storage, or during manufacture. Themethods of producing the temperature-activated gaseous precursor-filledvesicles may be carried out at a temperature below the boiling point ofthe gaseous precursor. In this embodiment, the gaseous precursor isentrapped within a vesicle such that the phase transition does not occurduring manufacture. Instead, the gaseous precursor-filled vesicles aremanufactured in the liquid phase of the gaseous precursor. Activation ofthe phase transition may take place at any time as the temperature isallowed to exceed the boiling point of the precursor. Also, knowing theamount of liquid in a droplet of liquid gaseous precursor, the size ofthe vesicles upon attaining the gaseous state may be determined.Alternatively, the gaseous precursors may be utilized to create stablegas filled vesicles which are preformed prior to use. In thisembodiment, the gaseous precursor is added to a container housing asuspending and/or stabilizing medium at a temperature below theliquid-gaseous phase transition temperature of the respective gaseousprecursor. As the temperature is then exceeded, and an emulsion isformed between the gaseous precursor and liquid solution, the gaseousprecursor undergoes transition from the liquid to the gaseous state. Asa result of this heating and gas formation, the gas displaces the air inthe head space above the liquid suspension so as to form gas filledlipid spheres which entrap the gas of the gaseous precursor, ambient gas(e.g., air) or coentrap gas state gaseous precursor and ambient air.This phase transition can be used for optimal mixing and stabilizationof the MRI contrast medium. For example, the gaseous precursor,perfluorobutane, can be entrapped in the biocompatible lipid or otherstabilizing compound, and as the temperature is raised, beyond 4° C.(boiling point of perfluorobutane) stabilizing compound entrappedfluorobutane gas results. As an additional example, the gaseousprecursor fluorobutane, can be suspended in an aqueous suspensioncontaining emulsifying and stabilizing agents such as glycerol orpropylene glycol and vortexed on a commercial vortexer. Vortexing iscommenced at a temperature low enough that the gaseous precursor isliquid and is continued as the temperature of the sample is raised pastthe phase transition temperature from the liquid to gaseous state. In sodoing, the precursor converts to the gaseous state during themicroemulsification process. In the presence of the appropriatestabilizing agents, surprisingly stable gas filled vesicles result.

[0161] Accordingly, the gaseous precursors may be selected to form a gasfilled vesicle in vivo or may be designed to produce the gas filledvesicle in situ, during the manufacturing process, on storage, or atsome time prior to use.

[0162] As a further embodiment of this invention, by pre-forming theliquid state of the gaseous precursor into an aqueous emulsion andmaintaining a known size, the maximum size of the microbubble may beestimated by using the ideal gas law, once the transition to the gaseousstate is effectuated. For the purpose of making gas filled vesicles fromgaseous precursors, the gas phase is assumed to form instantaneously andno gas in the newly formed vesicle has been depleted due to diffusioninto the liquid (generally aqueous in nature). Hence, from a knownliquid volume in the emulsion, one actually would be able to predict anupper limit to the size of the gaseous vesicle.

[0163] Pursuant to the present invention, an emulsion of a stabilizingcompound such as a lipid, and a gaseous precursor, containing liquiddroplets of defined size may be formulated, such that upon reaching aspecific temperature, the boiling point of the gaseous precursor, thedroplets will expand into gas filled vesicles of defined size. Thedefined size represents an upper limit to the actual size becausefactors such as gas diffusion into solution, loss of gas to theatmosphere, and the effects of increased pressure are factors for whichthe ideal gas law cannot account.

[0164] The ideal gas law and the equation for calculating the increasein volume of the gas bubbles on transition from the liquid to gaseousstates is as follows:

PV=nRT

[0165] where

[0166] P=pressure in atmospheres

[0167] V=volume in liters

[0168] n=moles of gas

[0169] T=temperature in ° K

[0170] R=ideal gas constant=22.4 L atmospheres deg⁻¹ mole⁻

[0171] With knowledge of volume, density, and temperature of the liquidin the emulsion of liquids, the amount (e.g., number of moles) of liquidprecursor as well as the volume of liquid precursor, a priori, may becalculated, which when converted to a gas, will expand into a vesicle ofknown volume. The calculated volume will reflect an upper limit to thesize of the gas filled vesicle, assuming instantaneous expansion into agas filled vesicle and negligible diffusion of the gas over the time ofthe expansion.

[0172] Thus, for stabilization of the precursor in the liquid state inan emulsion wherein the precursor droplet is spherical, the volume ofthe precursor droplet may be determined by the equation:

Volume (sphere)={fraction (4/3)}πr³

[0173] where

[0174] r=radius of the sphere

[0175] Thus, once the volume is predicted, and knowing the density ofthe liquid at the desired temperature, the amount of liquid (gaseousprecursor) in the droplet may be determined. In more descriptive terms,the following can be applied:

V _(gas)={fraction (4/3)}π(r _(gas))³

[0176] by the ideal gas law,

PV=nRT

[0177] substituting reveals,

V _(gas) =nRT/P _(gas)

[0178] or,

[0179] (A) n={fraction (4/3)} [πr_(gas) ³] P/RT

[0180] amount n={fraction (4/3)} [πr_(gas) ³ P/RT]*MW_(n)

[0181] Converting back to a liquid volume

[0182] (B) V_(liq)=[{fraction (4/3)} [πr_(gas) ³] P/RT]*MW_(n)/D]

[0183] where D=the density of the precursor

[0184] Solving for the diameter of the liquid droplet,

[0185] (C) diameter/2=[¾π [{fraction (4/3)}* [πr_(gas) ³] P/RT]MW_(n)/D]^(1/3)

[0186] which reduces to

[0187] Diameter=2[[r_(gas) ³] P/RT [MW_(n)/D]]^(1/3)

[0188] As a further means of preparing vesicles of the desired size foruse as MRI contrast agents in the present invention, and with aknowledge of the volume and especially the radius of the stabilizingcompound/precursor liquid droplets, one can use appropriately sizedfilters in order to size the gaseous precursor droplets to theappropriate diameter sphere.

[0189] A representative gaseous precursor may be used to form a vesicleof defined size, for example, 10μ diameter. In this example, the vesicleis formed in the bloodstream of a human being, thus the typicaltemperature would be 37° C. or 310° K. At a pressure of 1 atmosphere andusing the equation in (A), 7.54×10⁻¹⁷ moles of gaseous precursor wouldbe required to fill the volume of a 110 diameter vesicle.

[0190] Using the above calculated amount of gaseous precursor, and1-fluorobutane, which possesses a molecular weight of 76.11, a boilingpoint of 32.5° C. and a density of 0.7789 grams/mL⁻¹ at 20° C., furthercalculations predict that 5.74×10⁻¹⁵ grams of this precursor would berequired for a 10μ vesicle. Extrapolating further, and armed with theknowledge of the density, equation (B) further predicts that 8.47×10⁻¹⁶mLs of liquid precursor are necessary to form a vesicle with an upperlimit of 10μ.

[0191] Finally, using equation (C), an emulsion of lipid droplets with aradius of 0.0272μ or a corresponding diameter of 0.0544μ need be formedto make a gaseous precursor filled vesicle with an upper limit of a 10μvesicle.

[0192] An emulsion of this particular size could be easily achieved bythe use of an appropriately sized filter. In addition, as seen by thesize of the filter necessary to form gaseous precursor droplets ofdefined size, the size of the filter would also suffice to remove anypossible bacterial contaminants and, hence, can be used as a sterilefiltration as well.

[0193] This embodiment for preparing gas filled vesicles used assimultaneous magnetic resonance focused noninvasive ultrasound contrastagents in the methods of the present invention may be applied to allgaseous precursors activated by temperature. In fact, depression of thefreezing point of the solvent system allows the use gaseous precursorswhich would undergo liquid-to-gas phase transitions at temperaturesbelow 0° C. The solvent system can be selected to provide a medium forsuspension of the gaseous precursor. For example, 20% propylene glycolmiscible in buffered saline exhibits a freezing point depression wellbelow the freezing point of water alone. By increasing the amount ofpropylene glycol or adding materials such as sodium chloride, thefreezing point can be depressed even further. The selection ofappropriate solvent systems may be explained by physical methods aswell. When substances, solid or liquid, herein referred to as solutes,are dissolved in a solvent, such as water based buffers for example, thefreezing point is lowered by an amount that is dependent upon thecomposition of the solution. Thus, as defined by Wall, one can expressthe freezing point depression of the solvent by the following equation:

Inx _(a) =In(1−x _(b))=ΔH_(fus) /R(1/T _(o)−1/T)

[0194] where:

[0195] x_(a)=mole fraction of the solvent

[0196] x_(b)=mole fraction of the solute

[0197] ΔH_(fus)=heat of fusion of the solvent

[0198] T_(o)=Normal freezing point of the solvent

[0199] The normal freezing point of the solvent results from solving theequation. If x_(b) is small relative to x_(a), then the above equationmay be rewritten:

x _(b) =ΔH _(fus) /R[T−T _(o) /T _(o) T]≈ΔH _(fus) ΔT/RT _(o) ²

[0200] The above equation assumes the change in temperature ΔT is smallcompared to T₂. The above equation can be simplified further assumingthe concentration of the solute (in moles per thousand grams of solvent)can be expressed in terms of the molality, m. Thus,

X _(b) =m/[m+1000/ma]≈mMa/1000

[0201] where:

[0202] Ma=Molecular weight of the solvent, and

[0203] m=molality of the solute in moles per 1000 grams.

[0204] Thus, substituting for the fraction x_(b):

ΔT=[M _(a) RT _(o) ²/1000ΔH _(fus) ]m

or ΔT=K _(f) m, where

K _(f) =M _(a) RT _(o) ²/1000ΔH _(fus)

[0205] K_(f) is referred to as the molal freezing point and is equal to1.86 degrees per unit of molal concentration for water at one atmospherepressure. The above equation may be used to accurately determine themolal freezing point of gaseous-precursor filled vesicle solutions usedin the present invention.

[0206] Hence, the above equation can be applied to estimate freezingpoint depressions and to determine the appropriate concentrations ofliquid or solid solute necessary to depress the solvent freezingtemperature to an appropriate value.

[0207] Methods of preparing the temperature activated gaseousprecursor-filled vesicles include:

[0208] (a) vortexing an aqueous suspension of gaseous precursor-filledvesicles used in the present invention; variations on this methodinclude optionally autoclaving before shaking, optionally heating anaqueous suspension of gaseous precursor and lipid, optionally ventingthe vessel containing the suspension, optionally shaking or permittingthe gaseous precursor vesicles to form spontaneously and cooling downthe gaseous precursor filled vesicle suspension, and optionallyextruding an aqueous suspension of gaseous precursor and lipid through afilter of about 0.22μ, alternatively, filtering may be performed duringin vivo administration of the resulting vesicles such that a filter ofabout 0.22μ is employed;

[0209] (b) a microemulsification method whereby an aqueous suspension ofgaseous precursor-filled vesicles of the present invention areemulsified by agitation and heated to form vesicles prior toadministration to a patient; and

[0210] (c) forming a gaseous precursor in lipid suspension by heating,and/or agitation, whereby the less dense gaseous precursor-filledvesicles float to the top of the solution by expanding and displacingother vesicles in the vessel and venting the vessel to release air; and

[0211] (d) in any of the above methods, utilizing a sealed vessel tohold the aqueous suspension of gaseous precursor and stabilizingcompound such as biocompatible lipid, said suspension being maintainedat a temperature below the phase transition temperature of the gaseousprecursor, followed by autoclaving to move the temperature above thephase transition temperature, optionally with shaking, or permitting thegaseous precursor vesicles to form spontaneously, whereby the expandedgaseous precursor in the sealed vessel increases the pressure in saidvessel, and cooling down the gas filled vesicle suspension.

[0212] Freeze drying is useful to remove water and organic materialsfrom the stabilizing compounds prior to the shaking gas instillationmethod. Drying-gas instillation methods may be used to remove water fromvesicles. By pre-entrapping the gaseous precursor in the dried vesicles(i.e., prior to drying) after warning, the gaseous precursor may expandto fill the vesicle. Gaseous precursors can also be used to fill driedvesicles after they have been subjected to vacuum. As the dried vesiclesare kept at a temperature below their gel state to liquid crystallinetemperature, the drying chamber can be slowly filled with the gaseousprecursor in its gaseous state, e.g., perfluorobutane can be used tofill dried vesicles composed of dipalmitoylphosphatidylcholine (DPPC) attemperatures between 4° C. (the boiling point of perfluorobutane) andbelow 40° C., the phase transition temperature of the biocompatiblelipid. In this case, it would be most preferred to fill the vesicles ata temperature about 4° C. to about 5° C.

[0213] Preferred methods for preparing the temperature activated gaseousprecursor-filled vesicles comprise shaking an aqueous solution having astabilizing compound such as a biocompatible lipid in the presence of agaseous precursor at a temperature below the gel state to liquidcrystalline state phase transition temperature of the lipid. The presentinvention also contemplates the use of a method for preparing gaseousprecursor-filled vesicles comprising shaking an aqueous solutioncomprising a stabilizing compound such as a biocompatible lipid in thepresence of a gaseous precursor, and separating the resulting gaseousprecursor-filled vesicles for MRI imaging use.

[0214] Vesicles prepared by the foregoing methods are referred to hereinas gaseous precursor-filled vesicles prepared by a gel state shakinggaseous precursor instillation method.

[0215] Conventional, aqueous-filled liposomes of the prior art areroutinely formed at a temperature above the phase transition temperatureof the lipids used to make them, since they are more flexible and thususeful in biological systems in the liquid crystalline state. See, forexample, Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. 1978, 75,4194-4198. In contrast, the vesicles made according to preferredembodiments described herein are gaseous precursor-filled, which impartsgreater flexibility, since gaseous precursors after gas formation aremore compressible and compliant than an aqueous solution. Thus, thegaseous precursor-filled vesicles may be utilized in biological systemswhen formed at a temperature below the phase transition temperature ofthe lipid, even though the gel phase is more rigid.

[0216] The methods contemplated by the present invention provide forshaking an aqueous solution comprising a stabilizing compound such as abiocompatible lipid in the presence of a temperature activated gaseousprecursor. Shaking, as used herein, is defined as a motion that agitatesan aqueous solution such that gaseous precursor is introduced from thelocal ambient environment into the aqueous solution. Any type of motionthat agitates the aqueous solution and results in the introduction ofgaseous precursor may be used for the shaking. The shaking must be ofsufficient force to allow the formation of a suitable number of vesiclesafter a period of time. Preferably, the shaking is of sufficient forcesuch that vesicles are formed within a short period of time, such as 30minutes, and preferably within 20 minutes, and more preferably, within10 minutes. The shaking may be by microemulsifying, by microfluidizing,for example, swirling (such as by vortexing), side-to-side, or up anddown motion. In the case of the addition of gaseous precursor in theliquid state, sonication may be used in addition to the shaking methodsset forth above. Further, different types of motion may be combined.Also, the shaking may occur by shaking the container holding the aqueouslipid solution, or by shaking the aqueous solution within the containerwithout shaking the container itself. Further, the shaking may occurmanually or by machine. Mechanical shakers that may be used include, forexample, a shaker table, such as a VWR Scientific (Cerritos, Calif.)shaker table, a microfluidizer, Wig-L-Bug™ (Crescent DentalManufacturing, Inc., Lyons, Ill.), which has been found to giveparticularly good results, and a mechanical paint mixer, as well asother known machines. Another means for producing shaking includes theaction of gaseous precursor emitted under high velocity or pressure. Itwill also be understood that preferably, with a larger volume of aqueoussolution, the total amount of force will be correspondingly increased.Vigorous shaking is defined as at least about 60 shaking motions perminute, and is preferred. Vortexing at least 1000 revolutions perminute, an example of vigorous shaking, is more preferred. Vortexing at1800 revolutions per minute is most preferred.

[0217] The formation of gaseous precursor-filled vesicles upon shakingcan be detected by the presence of a foam on the top of the aqueoussolution. This is coupled with a decrease in the volume of the aqueoussolution upon the formation of foam. Preferably, the final volume of thefoam is at least about two times the initial volume of the aqueous lipidsolution; more preferably, the final volume of the foam is at leastabout three times the initial volume of the aqueous solution; even morepreferably, the final volume or the foam is at least about four timesthe initial volume of the aqueous solution; and most preferably, all ofthe aqueous lipid solution is converted to foam.

[0218] The required duration of shaking time may be determined bydetection of the formation of foam. For example, 10 ml of lipid solutionin a 50 ml centrifuge tube may be vortexed for approximately 15-20minutes or until the viscosity of the gaseous precursor-filled vesiclesbecomes sufficiently thick so that it no longer clings to the side wallsas it is swirled. At this time, the foam may cause the solutioncontaining the gaseous precursor-filled vesicles to raise to a level of30 to 35 ml.

[0219] The concentration of stabilizing compound, especially lipidrequired to form a preferred foam level will vary depending upon thetype of stabilizing compound such as biocompatible lipid used, and maybe readily determined by one skilled in the art, once armed with thepresent disclosure. For example, in preferred embodiments, theconcentration of 1,2-dipalimitoylphosphatidylcholine (DPPC) used to formgaseous precursor-filled vesicles according to methods contemplated bythe present invention is about 20 mg/ml to about 30 mg/ml salinesolution. The concentration of distearoylphosphatidylcholine (DSPC) usedin preferred embodiments is about 5 mg/ml to about 10 mg/ml salinesolution.

[0220] Specifically, DPPC in a concentration of 20 mg/ml to 30 mg/ml,upon shaking, yields a total suspension and entrapped gaseous precursorvolume four times greater than the suspension volume alone. DSPC in aconcentration of 10 mg/ml, upon shaking, yields a total volumecompletely devoid of any liquid suspension volume and contains entirelyfoam.

[0221] It will be understood by one skilled in the art, once armed withthe present disclosure, that the lipids and other stabilizing compoundsused as starting materials, or the vesicle final products, may bemanipulated prior and subsequent to being subjected to the methodscontemplated by the present invention. For example, the stabilizingcompound such as a biocompatible lipid may be hydrated and thenlyophilized, processed through freeze and thaw cycles, or simplyhydrated. In preferred embodiments, the lipid is hydrated and thenlyophilized, or hydrated, then processed through freeze and thaw cyclesand then lyophilized, prior to the formation of gaseous precursor-filledvesicles.

[0222] According to the methods contemplated by the present invention,the presence of gas, such as and not limited to air, may also beprovided by the local ambient atmosphere. The local ambient atmospheremay be the atmosphere within a sealed container, or in an unsealedcontainer, may be the external environment. Alternatively, for example,a gas may be injected into or otherwise added to the container havingthe aqueous lipid solution or into the aqueous lipid solution itself inorder to provide a gas other than air. Gases that are not heavier thanair may be added to a sealed container while gases heavier than air maybe added to a sealed or an unsealed container. Accordingly, the presentinvention includes co-entrapment of air and/or other gases along withgaseous precursors.

[0223] As already described above in the section dealing with thestabilizing compound, the preferred methods contemplated by the presentinvention are carried out at a temperature below the gel state to liquidcrystalline state phase transition temperature of the lipid employed. By“gel state to liquid crystalline state phase transition temperature”, itis meant the temperature at which a lipid bilayer will convert from agel state to a liquid crystalline state. See, for example, Chapman etal., J. Biol. Chem. 1974, 249, 2512-2521.

[0224] Hence, the stabilized vesicle precursors described above, can beused in the same manner as the other stabilized vesicles used in thepresent invention, once activated by application to the tissues of ahost, where such factors as temperature or pH may be used to causegeneration of the gas. It is preferred that this embodiment is onewherein the gaseous precursors undergo phase transitions from liquid togaseous states at near the normal body temperature of said host, and arethereby activated by the temperature of said host tissues so as toundergo transition to the gaseous phase therein. More preferably still,this method is one wherein the host tissue is human tissue having anormal temperature of about 37° C., and wherein the gaseous precursorsundergo phase transitions from liquid to gaseous states near 37° C.

[0225] All of the above embodiments involving preparations of thestabilized gas filled vesicles used in the present invention, may besterilized by autoclave or sterile filtration if these processes areperformed before either the gas instillation step or prior totemperature mediated gas conversion of the temperature sensitive gaseousprecursors within the suspension. Alternatively, one or moreanti-bactericidal agents and/or preservatives may be included in theformulation of the contrast medium, such as sodium benzoate, allquaternary ammonium salts, sodium azide, methyl paraben, propyl paraben,sorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylatedhydroxytoluene, chlorobutanol, dehydroacetic acid, ethylenediamine,monothioglycerol, potassium benzoate, potassium metabisulfite, potassiumsorbate, sodium bisulfite, sulfur dioxide, and organic mercurial salts.Such sterilization, which may also be achieved by other conventionalmeans, such as by irradiation, will be necessary where the stabilizedmicrospheres are used for imaging under invasive circumstances, e.g.,intravascularly or intraperitonealy. The appropriate means ofsterilization will be apparent to the artisan instructed by the presentdescription of the stabilized gas filled vesicles and their use. Thecontrast medium is generally stored as an aqueous suspension but in thecase of dried vesicles or dried lipidic spheres the contrast medium maybe stored as a dried powder ready to be reconstituted prior to use.

[0226] The invention is further demonstrated in the following propheticExamples 1-11. The examples, however, are not intended to in any waylimit the scope of the present invention.

EXAMPLES Example 1

[0227] Transferrin is coupled to dextran and this is added to a solutionof iron salts. The solution of superparamagnetic iron oxides is preparedby dissolving a mixture of ferrous and ferric iron salts in water andHCl at pH 1.0 in an anaerobic environment in the chamber of a HeatSystems Probe (Heat Systems, Farmingdale, N.Y.) sonicator equipped withan atmosphere and pressure chamber. The sonicator is activated using thestandard sized horn on medium/high power and as oxygen gas is bubbledthrough the solution the pH is raised suddenly to pH=12. The result isiron oxide nanoparticles composed of magnetite, Fe₃O₄. The nanoparticlesare washed in normal saline and differential centrifugation is used toharvest the nanoparticles with diameter of 20 nm and less. Thesenanoparticles are suspended in n-hexane at a concentration of 10 mg perml nanoparticles with 10 mg per ml dipalmitoylphospatidylcholine. Then-hexane is evaporated and the lipid coated iron oxide nanoparticles arelyophilized. Accordingly, iron oxide nanoparticles are prepared whichare coated with dextran bearing transferrin. The superparamagnetic ironoxide nanoparticles at a concentration of 10 mg per ml are mixed with 2mg per ml perfluoropentane and 20 mg per ml pluronic F-68 with 10 mg perml dioleoylphosphatidylcholine in sterile water with 5.5% by weightmannitol. This is microfluidized and results in a colloidal suspensionof perfluoropentane coated with transferrin labeled magnetite particles.This is administered i.v. (dose=5 ml) to a 25 year old female patientwith suspected ectopic pregnancy. The magnetically labeled vesicleslocalize in the ectopic pregnancy as the transferrin binds to the fetaltissue and this is visualized by MRI. High energy continuous waveultrasound, 2 MHz, 2.5 Watts/cm is applied to the ectopic fetal tissue.By virtue of increased absorption if sound energy caused by the vesiclesthe ectopic fetal tissue is then destroyed by the ultrasound energy.This avoids an open procedure such as laparotomy or a more invasiveprocedure such as laparoscopy as the therapeutic ultrasound undermagnetic resonance guidance can generally be performed transcutaneouslywithout having to gain surgical access.

Example 2

[0228] A colloidal suspension of perfluorohexane (0.2 mg per ml) andperfluorpentane (0.2 mg per ml) is prepared in 10 mg per ml phospholipid(82 mole percent DPPC, 7 mole percent DPPE-PEG 5000 and 10 mole percentDPPA and 1 mole % DPPE-PEG 5000-anti fibrin antibody) with 20 mg per mlpluronic F-68 and 5.5% by weight mannitol. To this is added 5 mg per mlof iron oxide nanoparticles and this material is microfluidized asdisclosed in preceding examples. This material is administered i.v. to apatient with suspected vascular thromobosis. A magnetometersuperconducting quantum inferometry device (SQUID), a type of magneticimaging, is scanned over the patient's body, localizing an apparentregion of increased magnetic susceptibility to the patient's ilialveins. The presence of clot is confirmed via ultrasound imaging. Highenergy ultrasound, 500 milliwatts per cm² is then applied to the regionsof clot to which the vesicles are bound. Sonication is performed underthe guidance of the SQUID or magnetometer. The ultrasound transducermight be equipped with a SQUID as a part of the transducer. As thesonication occurs, a change in the magnetic susceptibility is detectedby the magnetometer. The microvesicles cause increased absorption ofsonic energy; the liquid to gaseous phase conversion of theperflourohexane in the emulsion is readily visible on either ultrasoundor by magnetic imaging as the vesicles expand during the heat expansionprocess and this results in local lysis of the clot and noninvasivesurgical alleviation of the thrombosis.

Example 3

[0229] Gas filled microvesicles impregnated with paramagnetic iron oxideparticles in the microvesicle membrane are injected into the antecubitalfossa. The vesicles are preferential taken up by the lymphatic vesselswhich could be identified on magnetic resonance with a superconductingquantum inferometry device (SQUID) as clear outlines of tumor burdenednodes. Upon identification of the nodes, a focused ultrasound transduceris then aimed at the site of the identified tumor burden and acontinuous wave train of ultrasound is applied under SQUID guidance. Themicrovesicles are made to resonate and release energy in the form ofthermal energy, thereby heating and subsequently destroying the tumor.Upon re-imaging by magnetic imaging using the SQUID device, the image ofthe tumor burden in the lymphatic is no longer identifiable, indicatingdestruction of the tumor, thereby providing a noninvasive surgicaltechnique.

Example 4

[0230] Gas filled microvesicles prepared with manganeseN,N′-Bis-(carboxy-decylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N′-diacetate,(MN-EDTA-DDP), and Mn-EDTA-ODP, a paramagnetic complex disclosed in U.S.Pat. No. 5,312,617, the disclosure of which is incorporated herein byreference in its entirety, is injected into a patient with malignantmelanoma. The exact size and location of the tumor in the lymph chain isidentified on MRI and treated with real time simultaneous magneticresonance imaging and sonication using a magnetic resonance compatibletransducer operating at 1.5 Watts per cm² and a frequency of 0.75 MHz.The presence of the bubbles results in increased deposition of energy,heating of the tissue as well as local cavitation. The degree of tissuenecrosis as well as the temperature of the tissue is monitorednon-invasively by simultaneous real time magnetic resonance imaging on amachine equipped with echoplanar imaging gradients, thus effectingnoninvasive surgery.

Example 5

[0231] In a patient with a cerebral arteriovenous malformation (AVM), askull flap is created and the dura is surgically exposed. The patient isplaced in an MRT-0.5 Tesla, interventional magnetic resonance imagingsystem, (GE Medical Systems, Milwaukee, Wis.). This magnetic resonancesystem allows access to the patient during surgical procedures whilesimultaneous magnetic resonance imaging. The patient is injected with0.2 ml per kg of Aerosomes™ composed of 2 mg per ml of lipid containing75 mole % DPPC, 8 mole % DPPA and 8 mole % DPPE-PEG 5000 with 9 mole %Platelet-Activation Factor (PAF), Avanti Polar Lipids, Alabaster Alabamaentrapping a mixture of air and perfluorobutane gas. The purpose of thePAF is to activate platelets after release of the PAF from theAerosomes™ to stimulate thrombosis of the AVM. During magnetic resonanceimaging a high energy magnetic resonance compatible ultrasoundtransducer equipped with imaging and therapy functions is positionedover the AVM. After the Aerosomes™ are injected I.V. they pass throughthe large vessels and microcirculation supplying the AVM. Themicrobubbles are readily visualized by both magnetic resonance andultrasound. On magnetic resonance the bubbles are portrayed as signalvoids on gradient echo bright blood images obtained during transitthrough the AVM and on ultrasound as a snow-storm of specularreflections. The ultrasound transducer is focussed onto the AVM suchthat the focal zone of the ultrasound corresponds to the vascular nidus.Simultaneous magnetic resonance imaging and ultrasound are performed asthe power level on ultrasound is increased (e.g. up to several watts).While emissions from cavitation obscure visualization of much of theanatomy on ultrasound, magnetic resonance still shows the surroundingtissues with exquisite detail. The surgeon operating the high energyultrasound is therefore much better able to control the aiming, firingand energy levels of the high energy transducer and avoid damagingcritical surrounding cerebral vascular structures. The procedure resultsin ablation of the AVM, regions of coagulative necrosis as well asthrombosis of the vascular nidus. At the end of the procedure much ofthe anatomy is obscured on ultrasound due to the coagulative necrosisand collections of microbubbles within the tissue, but magneticresonance shows the entire surgical field as well as the surgicaleffects on the treated and surrounding tissues.

Example 6

[0232] A trauma victim with suspected hemorrhage is scanned by MRI. Thescan shows a hemorrhage from the spleen. Aerosomes as in the exampleabove except that the Aerosomes also entrap 1 mg per ml of thrombin areinjected I.V. and simultaneous magnetic resonance and ultrasound imagingare performed. As the Aerosomes pass through the splenic artery theultrasound power on the magnetic resonance compatible ultrasoundtransducer is increased by the surgeon to about 1.0 Watt per cm² and theAerosomes pop releasing PAF and thrombin. Thrombosis is achieved and thehemorrhage is stopped. Simultaneous magnetic resonance angiographyconfirms the thrombosis of the splenic artery. This minimally invasiveprocedure is cheaper and caused less morbidity than conventional opensurgery.

Example 7

[0233] 100 ml of perfluoropentane vesicles coated with phospholipid, 82mole % DPPC, 8 mole % DPPE-PEG 2000 and 10 mole % DPPA (meandiameter=about 1 micron) with 10 mole percent alkylated complexes ofmanganese (Mn-EDTA-ODP) bearing antibodies, Monoclonal Antibody toBreast Cancer (human) CA-15-3, IgG1, (SIGNET LABS, Dedham, Mass.) tohuman breast carcinoma is administered intravenously to a patient withbreast carcinoma. The vesicles also contain 10 mole percent alkylatedderivative of doxorubicin bound to the vesicle membranes. Four hourslater the patient is scanned via MRI. Enhancing lymph nodes areidentified in the axilla indicating metastatic disease. A magneticresonance compatible 1 MHz ultrasound probe is positioned over the lymphnodes and high energy continuous wave ultrasound at 200 mW/cm² isapplied to the lymph nodes. Simultaneous real time magnetic resonanceimaging is performed on a commercially available magnet, e.g. 1.5 Tesla(GE Signa, Milwaukee, Wis.), using rapid pulse sequences, e.g. SpoiledGRASS, TR=30 msec and TE=5 msec with a 30° flip angle.

[0234] As the vesicles expand during heating they are seen as anincreased region of low signal intensity on the magnetic resonanceimages corresponding to the zone of magnetic susceptibility caused bythe vesicles. As the vesicles “pop” this is seen as a transient regionof even more demonstrable hypointensity. Thereafter the vesiclesdisappear after they have “popped” and cleared. As the vesicles pop, thedoxorubicin pro-drug is released and activated in the neoplastic lymphnodes.

Example 8

[0235] Superparamagnetic iron oxides are prepared by dissolving amixture of ferrous and ferric iron salts in water and HCl at pH 1.0 inan anaerobic environment in the chamber of a Heat Systems Probe (HeatSystems, Farmingdale, N.Y.) sonicator equipped with an atmosphere andpressure chamber. The sonicator is activated using the standard sizedhorn on medium/high power and as oxygen gas is bubbled through thesolution the pH is raised suddenly to pH=12. The result is iron oxidenanoparticles composed of magnetite, Fe₃O₄. The nanoparticles are washedin normal saline and differential centrifugation is used to harvest thenanoparticles with diameter of 20 nm and less. These nanoparticles aresuspended in n-hexane at a concentration of 10 mg per ml nanoparticleswith 10 mg per ml dipalmitoylphospatidylcholine. The n-hexane isevaporated and the lipid coated iron oxide nanoparticles arelyophilized. The lipid coated iron oxide nanoparticles are then added10% by weight to 1 mg per ml of phospholipid composed of 82 mole %dipalmitoylphosphatidylcholine (DPPC) with 8 mole percentdipalmitylphosphatidylethanolamine-PEG 5,000 (DPPE-PEG 500) and 8 molepercent dipalmitoylphosphatidic acid (DPPA) with 2 mole percentpalmitoylated cis-platinum derivative. This mixture of lipids, lipidcoated iron oxides and palmitoylated pro-drug is suspended at a finallipid concentration of 1 mg per ml in normal saline in a sealed sterilecontainer with a head space of perfluorobutane gas. The material isshaken for 2 minutes on a Wig-L-Bug™ at 4,200 r.p.m and results in lipidcoated pro-drug bearing vesicles, the surfaces of which are studded withiron oxide nanoparticles. A dose of 20 ml of these vesicles (meandiameter=about 2 microns, bubble conc.=1×10⁹ per ml) is injectedintravenously into a patient and magnetic resonance imaging is performedobtaining rapid GRASS sequences. The iron oxide labeled vesicles areeasily seen on magnetic resonance as susceptibility agents causingregions of hypointensity. The pro-drug vesicle based contrast agent isshown to accumulate in regions of vascularized tumors involving thelymph nodes and other tissues. Ultrasound 1 MHz is applied as above to“pop” the vesicles under magnetic resonance guidance and achieve localdrug delivery.

Example 9

[0236] Anti-myosin antibody, anti-myosin antibody, Chicken Muscle,Catalogue No. 476123, (CAL BIOCHEM, La Jolla, Calif.) is coupled todipalmitolyphosphatidyl-ethanolamine. This is added 0.1 mg per ml to aconcentration of 0.1 mg per ml perfluoropentane and 5 mg per ml ofphospholipid (90 mole % DPPC with 10 mole % DPPA) in 5.5% by weightmannitol in sterile water. This mixture is bubbled with oxygen andoxygen-17 and then microemulsified using a microfluidizer(Microfluidics, Newton, Mass.) at 16,000 psi for 20 passes resulting inemulsified antibody bearing colloids of perfluoropentane. Ten cc of thismaterial is injected into a patient with suspected myocardial infarctionand magnetic resonance imaging is performed. A region of enhancedsusceptibility is shown in the area of infarcted myocardium on the rapidGRASS magnetic resonance images. A magnetic resonance compatiblecontinuous wave ultrasound transducer is positioned over the myocardiumand the myocardium is treated with 100 watts per cm² ultrasound energy.This causes the bubbles to pop and release oxygen locally into theischemic tissue.

Example 10

[0237] A vesicle is prepared as in Example 9, except that the cationiclipid DOTMA, N-[1(-2,3-dioleoyloxy)propyl]N,N-trimethylammonium chlorideis substituted for DPPA and the DPPC is substituted with DPPE. Theantibody bearing colloidal particles of perfluoropentane are thenprepared as above and loaded with Oxygen-17 gas within theperfluoropentane. Then 10 micrograms per ml of DNA encoding the gene forvascular endothelial growth factor (VEGF) is added and the suspension isvortexed at low power setting for 2 minutes at 4° C. Magnetic resonanceimaging is performed in a patient with suspected myocardial infarction.A region of low signal intensity is identified in the myocardium about30 minutes after administration of the contrast agent. An magneticresonance compatible ultra-sound transducer is focused on the region ofischemic/infarcted myocardium and the perfluoropentane microbubbles are“popped” under real time simultaneous magnetic resonance imaging using agradient echo echoplanar imaging technique in a magnetic resonancesystem equipped with resonating gradients, (Advanced NMR, Woburn,Mass.), retrofit 1.5 Tesla, (GE Signa System, Milwaukee, Wis.). Thisresults in local integration of the gene for VEGF within theinfarcted/ischemic myocardium and local gene expression. New bloodvessels proliferate in response to the VEGF and there is increasedgrowth of new blood vessels. This results in healthier regionalmyocardium.

Example 11

[0238] Cationic vesicles are prepared by shaking a mixture of lipid 82mole % DPPC with 7 mole % DPPE-PEG 5,000 with 5 mole % DPPA and 6 mole %DOTMA with a head space of gas prepared from a mixture ofperfluorobutane and oxygen-16. After shaking, as disclosed in precedingexamples with the Wig-L-Bug™, 100 micrograms per ml of DNA for the VEGFgene is added and the mixture is gently vortexed for 1 minute. Thisresults in absorption of the DNA onto the surface of the microvesicles.A somewhat higher amount of vesicles is necessary for visualizationunder magnetic resonance than the magnetically labeled vesicles but thevesicles are still detectable on T2 weighted spin echo or fast spin echoor gradient echo pulse sequences. Magnetic resonance angiography (MRA),a type of magnetic resonance imaging, using a 2D Time of Flight pulsesequence is performed an a diabetic patient with peripheral vasculardisease involving the lower extremities. A significant stenosis is shownin the popliteal artery. The microvesicles bearing DNA are administeredi.v. and on the basis of the vesicle signals from MRI and ultrasound,high energy ultrasound is applied under magnetic resonance imagingguidance from the skin surface to focus energy on the region of stenosisin the popliteal artery. This results in localized bubble rupture andgene release at the site of arterial stenosis. Localized expression ofVEGF encourages collateral and new vessel formation improving blood flowto the distal leg.

[0239] The disclosures of each patent, patent application andpublication cited or described in this document are hereby incorporatedherein by reference, in their entirety.

[0240] Various modification of the invention, in addition to thosedescribed herein, will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. A non-invasive method for performing surgery inthe vasculature of a patient, said method comprising: (a) administeringintravascularly to said patient a composition comprising, in an aqueousdiluent, stabilized gas or gaseous precursor filled vesicles; and (b)applying ultrasound to said patient in an amount sufficient to induceactivation or rupture of said vesicles.
 2. A method according to claim 1wherein said activation or rupture of said vesicles results in thedisruption or destruction of tissue within the vasculature of saidpatient.
 3. A method according to claim 2 further comprising the step ofscanning the patient with diagnostic imaging to ascertain the presenceof said vesicles adjacent to said tissue.
 4. A method according to claim3 wherein said diagnostic imaging comprises magnetic resonance imaging.5. A method according to claim 2 wherein said tissue comprises athrombus.
 6. A method according to claim 5 wherein said surgeryalleviates a thrombosis in said patient.
 7. A method according to claim2 wherein said surgery repairs an aperture in said vasculature.
 8. Amethod according to claim 1 wherein said vesicles further comprise atargeting ligand.
 9. A method according to claim 1 wherein said vesiclesfurther comprise a therapeutic agent which is released upon applicationof said ultrasound.
 10. A method according to claim 1 wherein said gasor gaseous precursor comprises a fluorinated compound.
 11. A methodaccording to claim 10 wherein said fluorinated compound is selected fromthe group consisting of perfluorocarbons and sulfur hexafluoride.
 12. Amethod according to claim 11 wherein said fluorinated compound is aperfluorocarbon selected from the group consisting of perfluoropropane,perfluorobutane, perfluorocyclobutane, perfluoromethane,perfluoroethane, perfluorohexane, and perfluoropentane.
 13. A methodaccording to claim 12 wherein said fluorinated compound isperfluoropropane.
 14. A method according to claim 12 wherein saidfluorinated compound is perfluorobutane.
 15. A method according to claim1 wherein said vesicles comprise liposomes.
 16. A method according toclaim 15 wherein said liposomes comprise crosslinked or polymerizedlipids.
 17. A method according to claim 15 wherein said liposomescomprise a phospholipid.
 18. A method according to claim 15 wherein saidliposomes further comprise polyethylene glycol.
 19. A method accordingto claim 15 wherein said liposomes comprise a monolayer.
 20. A methodaccording to claim 1 wherein said vesicles comprise a polysaccharide.21. A method according to claim 20 wherein said polysaccharide comprisesgalactose.
 22. A method according to claim 1 wherein said vesiclescomprise a polymer.
 23. A method according to claim 22 wherein saidpolymer comprises a methacrylate.
 24. A method according to claim 1wherein said vesicles comprise a surfactant.
 25. A method according toclaim 24 wherein said vesicles have been rehydrated from lyophilizedvesicles.
 26. A method according to claim 1 wherein said vesiclescomprise a protein.
 27. A method according to claim 26 wherein saidprotein comprised albumin.
 28. A method according to claim 4 whereinsaid composition further comprises a paramagnetic agent.
 29. A methodaccording to claim 28 wherein the paramagnetic agent comprises aparamagnetic ion selected from the group consisting of transition,lanthanide and actinide elements.
 30. A method according to claim 29wherein the paramagnetic ion is selected from the group consisting ofGd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II),Ni(II), Eu(III) and Dy(III).
 31. A method according to claim 30 whereinthe paramagnetic ion is Mn(II).
 32. A method according to claim 28wherein the paramagnetic agent comprises a nitroxide.
 33. A methodaccording to claim 4 wherein said composition comprises asuperparamagnetic agent.
 34. A method according to claim 33 wherein thesuperparamagnetic agent comprises a metal oxide or metal sulfide.
 35. Amethod according to claim 34 wherein the superparamagnetic agentcomprises a metal oxide wherein the metal is iron.
 36. A methodaccording to claim 35 wherein the superparamagnetic agent is ferritin,iron, magnetic iron oxide, γ-Fe₂O₃, manganese ferrite, cobalt ferriteand nickel ferrite.
 37. A method according to claim 4 wherein saidvesicles are filled with ¹⁹F and said magnetic resonance imaging isnuclear magnetic resonance.
 38. A method according claim 4 wherein saidvesicles are filled with a gas selected from the group consisting ofrubidium enhanced xenon, rubidium enhanced argon, rubidium enhancedhelium, and rubidium enhanced neon.
 39. A method according to claim 1wherein said vesicles have a mean diameter less than about 30 microns.40. A method according to claim 39 wherein said vesicles have a meandiameter less than about 12 microns.
 41. A non-invasive method for thedisruption of tissue in the vasculature of a patient, said methodcomprising: (a) administering intravascularly to said patient acomposition comprising, in an aqueous diluent, stabilized gas or gaseousprecursor filled vesicles; (b) scanning the patient with diagnosticimaging to ascertain the presence of said vesicles adjacent to saidtissue; and (c) applying ultrasound to said patient in an amountsufficient to induce activation or rupture of said vesicles adjacent tosaid tissue.
 42. A method according to claim 41 wherein said methodresults in the alleviation of diseased tissue in said vasculature.
 43. Amethod according to claim 42 wherein said method results in thealleviation of a thrombus in said vasculature.
 44. A method according toclaim 41 wherein said diagnostic imaging comprises magnetic resonanceimaging.
 45. A method according to claim 41 wherein said vesiclesfurther comprise a targeting ligand.
 46. A method according to claim 41wherein said vesicles further comprise a therapeutic agent which isreleased upon application of said ultrasound.
 47. A method according toclaim 41 wherein said gas or gaseous precursor comprises a fluorinatedcompound.
 48. A method according to claim 47 wherein said fluorinatedcompound is selected from the group consisting of perfluorocarbons andsulfur hexafluoride.
 49. A method according to claim 48 wherein saidfluorinated compound is a perfluorocarbon selected from the groupconsisting of perfluoropropane, perfluorobutane, perfluorocyclobutane,perfluoromethane, perfluoroethane, perfluorohexane, andperfluoropentane.
 50. A method according to claim 49 wherein saidfluorinated compound is perfluoropropane.
 51. A method according toclaim 49 wherein said fluorinated compound is perfluorobutane.
 52. Amethod according to claim 41 wherein said vesicles comprise liposomes.53. A method according to claim 52 wherein said liposomes comprisecrosslinked or polymerized lipids.
 54. A method according to claim 52wherein said liposomes comprise a phospholipid.
 55. A method accordingto claim 52 wherein said liposomes further comprise polyethylene glycol.56. A method according to claim 52 wherein said liposomes comprise amonolayer.
 57. A method according to claim 41 wherein said vesiclescomprise a polysaccharide.
 58. A method according to claim 57 whereinsaid polysaccharide comprises galactose.
 59. A method according to claim41 wherein said vesicles comprise a polymer.
 60. A method according toclaim 59 wherein said polymer comprises a methacrylate.
 61. A methodaccording to claim 41 wherein said vesicles comprise a surfactant.
 62. Amethod according to claim 61 wherein said vesicles have been rehydratedfrom lyophilized vesicles.
 63. A method according to claim 41 whereinsaid vesicles comprise a protein.
 64. A method according to claim 62wherein said protein comprised albumin.
 65. A method according to claim44 wherein said composition further comprises a paramagnetic agent. 66.A method according to claim 65 wherein the paramagnetic agent comprisesa paramagnetic ion selected from the group consisting of transition,lanthanide and actinide elements.
 67. A method according to claim 66wherein the paramagnetic ion is selected from the group consisting ofGd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II),Ni(II), Eu(III) and Dy(III).
 68. A method according to claim 67 whereinthe paramagnetic ion is Mn(II).
 69. A method according to claim 66wherein the paramagnetic agent comprises a nitroxide.
 70. A methodaccording to claim 44 wherein said composition comprises asuperparamagnetic agent.
 71. A method according to claim 70 wherein thesuperparamagnetic agent comprises a metal oxide or metal sulfide.
 72. Amethod according to claim 71 wherein the superparamagnetic agentcomprises a metal oxide wherein the metal is iron.
 73. A methodaccording to claim 72 wherein the superparamagnetic agent is ferritin,iron, magnetic iron oxide, γ-Fe₂O₃, manganese ferrite, cobalt ferriteand nickel ferrite.
 74. A method according to claim 44 wherein saidvesicles are filled with ¹⁹F and said magnetic resonance imaging isnuclear magnetic resonance.
 75. A method according claim 44 wherein saidvesicles are filled with a gas selected from the group consisting ofrubidium enhanced xenon, rubidium enhanced argon, rubidium enhancedhelium, and rubidium enhanced neon.
 76. A method according to claim 41wherein said vesicles have a mean diameter less than about 30 microns.77. A method according to claim 76 wherein said vesicles have a meandiameter less than about 12 microns.